Compounds for treatment of heart failure

ABSTRACT

A combination of: a first tetracycline (TC) component; and a second component capable of releasing nitric oxide (NO) or a nitrate capable of mimicking NO effects in vivo (NO mimetic). The combinations of the invention advantageously act as more effective MMP modulators with selective reductions in circulating MMP-9 levels in-vivo and inhibitory effects on MMP-2 and MMP-9 levels in-vitro. The combinations of the invention also advantageously act as modulators of inflammation mediators. The co-existence of abnormalities of MMP enzymes and inflammation in many diseases make these characteristics advantageous. Therefore, the various combinations of the invention find utility in medical applications where MMPs and/or inflammation is implicated.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a national stage application under 35 U.S.C.§371 of International Application No. PCT/EP2011/072243, filed Dec. 8,2011, which in turn claims priority to United Kingdom Patent ApplicationNo. 1020811.4, filed Dec. 8, 2010, the contents of each of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a compound comprising a tetracycline,and an associated molecule that is capable of releasing nitric oxide(NO). Also disclosed are methods for the preparation of such compounds,and their use in treating or preventing heart failure, optionally heartfailure caused by or associated with diastolic dysfunction.

BACKGROUND TO THE INVENTION

The prevalence of heart failure (HF) is increasing in the developedworld and the cost of providing medical care for an expanding HFpopulation imposes an increasingly heavy burden on healthcare systemsthroughout the world. Most commonly, HF is associated with impaired leftventricular (LV) systolic function. However, at least half of allpatients with typical symptoms of congestive HF have a normal orslightly reduced left ventricular ejection fraction (LVEF) (>50%). Thepredominant cause of heart failure with preserved ejection fraction(HFpEF) is diastolic heart failure (DHF). Heart failure (HF) withpreserved ejection fraction (HFpEF) is predominantly caused byhypertension, is often preceded by asymptomatic left ventriculardiastolic dysfunction (ALVDD) and has few defined therapies. Thepredominant aetiological cause of DHF is myocardial fibrosis as a resultof long standing hypertension and metabolic abnormalities associatedwith diabetes and obesity. The rising prevalence of metabolic diseasedue to the obesity and diabetic epidemics means that DHF is a majorpublic health problem. DHF, similar to systolic HF has a five-yearmortality rate of 65%. In many of these patients, diastolic dysfunctioncaused by hypertensive heart disease (HHD) is implicated as a majorcontributor, if not a primary cause. Furthermore, the prevalence ofasymptomatic diastolic dysfunction in the community is significant withapproximately 25-30% of individuals >45 years of age being affected.There are no proven, life-saving therapies for treating DHF. Many of thewell-established drug therapies for systolic heart failure have beendirected at DHF without success. The diagnosis of DHF can present achallenge in routine clinical practice. The major limitation in thediagnosis of DHF is the identification of diastolic dysfunction (DD),which at present is predominantly reliant on Doppler echocardiographicstudies. Echocardiography has been used for many years to providestructural correlates to the clinical picture of HF. It can also measuremultiple clinically important parameters of cardiac function, includinghemodynamic status and LVEF, volumes and mass. The pathophysiology ofDHF includes delayed relaxation, impaired LV filling and/or increasedstiffness. These conditions result in an upward displacement of thediastolic pressure-volume relationship with increased end-diastolic,left atrial and pulmo-capillary wedge pressure leading to symptoms ofpulmonary congestion. Diagnosis of DHF requires three conditions; (1)presence of signs or symptoms of HF; (2) presence of normal or slightlyreduced LVEF (>50%) and (3) presence of increased diastolic fillingpressure. Data indicate that the underlying pathophysiology in diastolicdysfunction and DHF is related to myocardial interstitial disease.Collagen is a stable protein and its balanced turnover is estimated tobe 80-120 days. Alteration of collagen turnover by various mechanismscan lead to adverse accumulation of collagen in the myocardialinterstitium leading to fibrosis, increased tissue stiffness, reducedmyocardial compliance and impaired diastolic function. The successfulneurohumoral-based approach to pharmacotherapy in HF with systolicdysfunction has not resulted in similarly impressive results in HFpEF,implicating additional pathophysiological signals. Changes in theextracellular matrix (ECM), known as myocardial remodeling, are centralabnormalities in many patients with HFpEF and are characterized byinflammation, increased ECM turnover and myocardial fibrosis. Keymediators of inflammation are pro-inflammatory cytokines includinginterleukins (IL) (IL-1β, IL-6, IL-8) and tumor necrosis factor (TNF)α.Key regulators of the turnover of collagen and extracellular matrix(ECM) in the myocardium are the matrix metalloproteinases (MMPs) andtheir tissue inhibitors (TIMPs). MMPs in particular have been found toplay an important role in both inflammation and fibrosis. MMPs alsocontribute to collagen degradation and remodeling of the ECM aftermyocardial infarction. ECM turnover is regulated by matrixmetalloproteinases (MMPs), especially the “gelatinases”, MMP-2 andMMP-9, and their tissue inhibitors (TIMPs). MMP-2 and MMP-9 knockoutmodels are associated with reduced aortic elastin degradation andprotection from pressure overload hypertrophy, fibrosis and dysfunction.In the clinic, independent associations between ALVDD and HFpEF havebeen identified with markers of inflammation, fibrosis and MMP-9. Duringischemic cardiomyopathy, neutrophil proteinase activates latentmyocardial MMP, which can degrade the ECM. If unchecked by TIMPs, theECM continuously degrades, leading to ventricular dilatation anddiastolic dysfunction. Despite the emerging awareness of the potentialrole of collagen metabolism in the pathogenesis of diastolic HF thereare as yet no effective therapies for this form of HF. Pharmacologicalmodulation of MMPs may present an opportunity. However, all MMPsynthetic inhibitors developed to date have either been ineffective ordemonstrated dose- and duration-dependent drug-related side-effects,most which were musculoskeletal-related. Despite some promising animalstudies of MMP inhibitors showing attenuation of cardiovascularremodeling in chronic pressure-overload models, the approach of directinhibition of MMP enzymes has proven too toxic or ineffective in theclinic. An alternative approach in cardiovascular disease would inhibitproduction and/or secretion of inducible myocardial MMP-9. As well asclassic inflammatory diseases such as rheumatoid arthritis, hay fever,periodontitis, inflammation plays an important role in the developmentand progression of diabetes and a variety of cardiovascular conditions,most notably coronary atherosclerosis and congestive heart failure. Theterm “Diabetic cardiomyopathy” was coined 4 decades ago and describes a“silent, stiffening” of the heart tissue which can lead to heartfailure. There are no symptoms until heart failure occurs. It is presentin half of people with diabetes and is more prevalent thanwell-recognised “silent pumping problem” which has good treatmentavailable. This silent stiffening of the heart is linked to overweight,diabetes, high blood pressure and there are no specific therapies. Overthe past 20 years, basic and human research has shown that enzymes inthe heart called matrix metallproteinases or MMPs are involved in thestiffening process. They also affect large and small blood vessels andcause eye and kidney damage in diabetes. For example, in patients withdiabetic retinopathy, increased MMP-9 activity was observed in retinalmicrovessels and MMP-9 knockout was protective (Kowluru et al,Abrogation of MMP-9 Gene Protects Against the Development of Retinopathyin Diabetic Mice by Preventing Mitochondrial Damage. Diabetes. 2011 Sep.20 [Epub ahead of print]). Increased urinary excretion of MMP-9 inpatients supports a role for MMP-9 dysregulation in diabetic renaldysfunction (Thrailkill et al., Endocrine. 2010 April; 37(2):336-43).Aortic and coronary arteries of diabetic patients taken at autopsy hadhigher expression of MMP-9 compared to non-diabetics and were correlatedwith HbA1c as well as apoptosis (Ishibashi et al., J Atheroscler Thromb.2010 Jun. 30; 17(6):578-89). Elevated MMP-9 has also been associatedwith arterial stiffness in patients with diabetes (Chung et al.,Cardiovasc Res. 2009 Dec. 1; 84(3):494-504). Furthermore, human geneticpolymorphisms associated with MMP-9 elevation support a role for thisenzyme in the pathophysiology of vascular disease. The 1562C>T singlenucleotide polymorphism (SNP), which affects the promoter region ofMMP-9 gene and increases circulating levels of MMP-9, is significantlyassociated with vascular disease in type 2 diabetes mellitus (Wang etal., Biochem Biophys Res Commun. 2010 Jan. 1; 391(1):113-7). In age andsex matched controls, patients with type 2 diabetes without and withmicroangiopathy, T allele frequencies were 11.9%, 13.1% and 24.4%respectively (p<0.05). Similarly, in a cohort of asymptomatichypertensive patients, the 1562C>1 polymorphism is associated withincreased T allele frequency, higher plasma MMP-9 and evidence ofincreased hypertension and vascular stiffness, measured by pulse wavevelocity (Zhou et al. J Hum Hypertens. 2007 November; 21(11):861-7).Inflammation is also involved in the development and progression of somecancers (e.g., gallbladder carcinoma). Inflammation is mediated by acomplex interplay of mediators such as IL-1 beta, IL-4 and IL-8. IL-1beta induces COX-2, which causes brain levels of prostaglandin (PG)E2 torise, thus activating the thermoregulatory center for fever production.In the periphery, IL-1 beta activates IL-1 receptors on the endothelium,resulting in expression of adhesion molecules and chemokines, whichfacilitate the emigration of neutrophils into the tissue spaces. IL-1 ispro-inflammatory and has been implicated in various pro-inflammatorydiseases such as coronary atherosclerosis and congestive heart failureas well as diabetes where recent studies from animals, in-vitro culturesand clinical trials provide evidence that support a causative role forIL-1β as the primary agonist in the loss of beta-cell mass in type 2diabetes. IL-4 is a TH2 type anti-inflammatory and profibrosis cytokinethat stimulates and amplifies the inflammatory response by activation ofthe synthesis of types I and II collagen by fibroblasts and thepromotion of the progression of fibrosis. IL-4 also inhibits theproinflammatory response of TNF-α, IL-1 and IL-6. IL-4 stimulatesinflammatory responses, activates collagen synthesis, promotes fibrosisprogression, and inhibits the production of inflammatory cytokines. Thepatients with CHF had higher IL-4 and PIIINP values than the controls.Comparison of the IL-4 values between the patients and controls showed asignificantly greater difference in the CHF patients (12 [12] vs 4 [3]pg/mL; P<0.0001). Recent studies have shown that pro-inflammatorycytokines play a significant contributory role in the pathogenesis ofacute heart failure. The purpose of this study was to determine whetherthe serum IL-8 concentration in patients with acute myocardialinfarction (AMI), who were undergoing percutaneous coronary intervention(PCI) was related to the subsequent presence or absence of heartfailure. A study by Dominguez-Rodriguez 2006, included 50 patients whounderwent successful PCI. During their subsequent stay in the coronarycare unit, their maximum degree of heart failure was recorded. Serumlevels of IL-8 in patients more severe symptoms (Killip class >I) weresignificantly higher than those of with less severe symptoms (Killipclass I) (P<0.001). By multivariante analysis a higher level of IL-8 wasa significant predictor of heart failure after PCI. Similarly in HF, thepresence of the metabolic syndrome which puts patients at higher risk,plasma levels of IL-8 (p<0.05) were significantly higher in HF patientswith MetS than those without MetS.

Tetracyclines, commonly known for their broad-spectrum antimicrobialproperties, have been characterized as pleiotropic immunomodulatoryagents. In human studies, sub-antimicrobial doses of the tetracycline,doxycycline, have exerted potentially beneficial effects on inflammationthat could promote plaque stability in an effort to prevent acutecoronary syndrome, as doxycycline therapy has been shown to lead to apowerful reduction of aneurysmal wall neutrophil and cytotoxic T-cellcount; two cell types considered crucial for the process of aneurysmformation. Attempts have been made to attenuate MMP expression toinhibit aortic abdominal aneurysm formation using doxycycline, therebyreducing the need for surgery. Doxycycline has been shown to inhibitsecretion of MMP-2 and MMP-9 and is the only drug currently licensed forhuman use that relies on MMP inhibition. It is currently underevaluation in ALVDD and HF patients in our group for its effects oninflammation, MMPs, myocardial structure and function using cardiac MRI[EudraCT number: 2010-021664-16]. However, in several animal and humanstudies, the efficacy of MMP inhibition with doxycycline has beenquestioned. This may reflect non-specific inhibition of the wider MMPfamily with high doses and/or chronic therapy, involving inhibition ofboth constitutive and inducible enzymes. It prompted our group to createanalogues of doxycycline that target over-expression of inducible MMP-9rather than direct enzyme inhibition as a more effective and saferapproach. Evidence is emerging that members of the MMP and/or Adisintegrin and metalloproteinase (ADAM) family can serve not only aspotential markers for diagnosis and prognosis, early detection, and riskassessment, but also as indicators of tumor recurrence, metastaticspread, and response to primary and adjuvant therapy for breast cancer.MMP-9 levels in tumor tissue as well as serum, plasma, and urine aresignificantly elevated in patients with breast cancer. Recently, effortshave focused on the use of MMPs and ADAMs as potential biomarkers ofearly breast cancer. Studies indicate that urinary MMP-9 and ADAM12, inaddition to being predictive markers for breast cancer, may also proveuseful as noninvasive breast cancer risk assessment tools. Severalindependent studies have used circulating MMP-9 activity to predictmetastatic spread of disease as well as to monitor patient response toprimary and adjuvant therapy and to evaluate outcome. High levels ofserum MMP-9 and TIMP-1 are associated with increased incidence of lymphnode metastasis and decreased relapse-free and overall survival rates.MMPs may also be useful in predicting therapeutic efficacy. Plasma MMP-9levels decrease after the surgical removal of primary breast tumors anda progressive decrease in plasma MMP-9 was observed in patients whoresponded well to adjuvant therapy. Importantly, in all patients whosuffered a relapse of disease there was a gradual increase of plasmaMMP-9 activity 1 to 8 months before the clinical diagnosis ofrecurrence. Serum and tissue levels of MMP-9 are significantly higher inpatients with pancreatic ductal adenocarcinoma than in patients withchronic pancreatitis and healthy controls. Active MMP-2 levels areupregulated in the pancreatic juice of patients with cancer (100%) ascompared with patients with chronic pancreatitis (2%) or normal controls(0%). Several studies have reported that plasma and/or serum levels ofMMP-9 and TIMP-1 are elevated in patients with stage III or IV lungcancer when compared with those in patients with nonmalignant lungdiseases. Urinary MMP-2 and MMP-9 levels correlate with presence ofbladder cancer as well as stage and grade of disease. Several MMPspecies have been reported in urine from patients with primary tumors inthe bladder and prostate including MMP-2, MMP-9, MMP-9/neutrophilgelatinase-associated lipocalin complex and MMP-9 dimer. Each urinaryMMP species was detected at significantly higher rates in urine frompatients with cancer as compared with controls. The difference indetection of MMP species in the urine of the two types of cancersstudied may serve as a tumor-specific fingerprint that can indicate boththe presence of a tumor as well as its location. Increased levels ofMMP-9 and MMP-2 in urine correlate with increased expression of theseproteases in bladder tumor tissue as well. Urinary MMP-9 levels whencombined with telomerase analysis of exfoliated cells from voided urinecould also increase the sensitivity of cytology, a commonly used methodfor bladder cancer detection and monitoring. MMP-2 and MMP-9 have beenstudied as potential prognostic biomarkers of colorectal cancer.Enhanced MMP-9 staining in primary tumors was found to be an independentmarker of poor prognosis in a study with T3-T4 node-negative patients.Plasma MMP-2 and MMP-9 levels were significantly elevated in patientswith colorectal cancer and those with adenomatous polyps, andsignificant reduction in both were observed after tumor resections,suggesting their potential as markers for therapeutic efficacy. TheseMMPs may not be prognostic markers for tumor recurrence, however, sinceplasma proMMP-2 and -9 activities did not correlate with disease relapseafter surgery. Tutton and colleagues investigated whether plasma MMP-2and MMP-9 levels could be used as a surrogate for tumour expression incolorectal cancer patients and they found significant correlationsbetween plasma levels and tumor pre- and post-op. MMP-2, -9, and -14 areamong the most studied MMPs as biomarkers for ovarian cancer. MMP-9activity in tissue extracts was significantly increased in advancedovarian cancers (International Federation of Gynecology and Obstetricsstage III) compared with benign tumors and was found to be anindependent prognosticator of poor survival. In another study ofinvasive epithelial ovarian cancer, high stromal expressions of MMP-9and -14 were significantly correlated with cancer progression and wereindependent prognostic markers. Tissue MMPs have also been shown todistinguish different histotypes of ovarian cancer, which is asignificant finding given that different histotypes have differentprognoses. A recent study showed that more than 90% of clear-cellcarcinomas expressed moderate to high levels of MMP-2 or MMP-14,compared with 30% to 55% of the other ovarian cancer histotypes (serous,endometroid, and mucinous), whereas MMP-9 was expressed more widely inother histotypes. Importantly, the cellular source of MMPs must beconsidered when evaluating MMPs as ovarian cancer biomarkers. Forexample, strong MMP-9 levels in cancer cells were associated with longersurvival whereas strong stromal MMP-9 was associated with shortersurvival, suggesting a dual role for MMP-9 during ovarian cancerprogression. MMP-2, -9, -15, and -26 expression in tissue or serum havebeen positively correlated with Gleason score in prostate cancer. Amongthese MMPs, the activities of plasma MMP-2 and -9 increasedsignificantly in metastatic prostate cancer. Analysis of MMP-2 and -9levels in radical prostatectomy specimens revealed these two assignificant predictors of cancer recurrence. These two enzymes may alsobe markers of therapeutic efficacy, since both the levels and activitiesof plasma MMP-2 and -9 decreased significantly in metastatic patientsafter therapy. In addition, increased urinary MMP-9 activity has beenshown to distinguish between prostate and other types of cancer (e.g.bladder cancer). MMPs can also be combined with other markers toincrease their predictive capability. For example, the mRNA ratio ofgelatinases to E-cadherin in biopsy samples independently predictedprostate cancer stage. Elevated tissue levels of MMP-2 and MMP-9 havebeen reported in aggressive brain tumors. Both latent and activatedforms of MMP-2 and MMP-9 have been detected in the cerebrospinal fluidof patients with brain tumors. In studies of primary glial tumors andother central nervous system tumors, we have recently shown thatdetection of MMP-2, MMP-9, MMP-9/neutrophil gelatinase-associatedlipocalin complex, and/or vascular endothelial growth factor in theurine predicted disease status and therapeutic efficiency of patientswith brain cancer. Importantly, these studies showed that theupregulation of MMP-2 and -9 in the source tumor tissue was alsoreflected in CSF as well as in urine of these patients. Tumor cellsoverexpress proteases and/or induce expression of these enzymes inneighboring stromal cells in order to degrade the basement membrane andinvade the surrounding tissue. Several MMPs have been implicated in theECM degradation associated with tumor growth and angiogenesis. Thisproteolytic activity is also required for a cancer cell to invade anearby blood vessel (intravasation) and then extravasate at a distantlocation and invade the distant tissue in order to seed a new metastaticsite. MMPs have been shown to promote angiogenesis through their releaseof angiogenic factors stored in the ECM such as vascular endothelialgrowth factor (VEGF) and basic fibroblast growth factor (bFGF; 3).Stroma-derived MMP-9 can facilitate the liberation of ECM-sequesteredVEGF during tumor angiogenesis. MMPs play complex and sometimesconflicting roles in regulating angiogenesis. Remodeling of the ECMduring angiogenesis is accomplished largely through the activity ofMMPs. Angiogenic mitogens, such as bFGF and VEGF, can stimulate theproduction of MMPs by capillary endothelial cells. Studies have alsodemonstrated that MMPs are involved in the angiogenic switch, one of theearliest stages of tumor growth and progression. It has been shown thatMMP-9 can be a regulator of the angiogenic switch in a pancreatic tumormodel, further confirming the pro-angiogenic role of MMPs. Thesefindings strongly suggest that MMP activity is critical, not only to theinitiation of angiogenesis, but to the maintenance of the growingvascular bed, which in turn supports tumor growth and metastasis. MMPactivity can, however, result in the production of negative regulatorsof angiogenesis as well. ECM degradation products display uniquebiologic properties that can trigger a variety of cellular signals. MMPshave also been implicated in the epithelial to mesenchymal transition(EMT), a hallmark of cancer progression to metastasis. Activation ofgrowth factors and cleavage of adhesion molecules are some of theproposed mechanisms underlying MMP-induced EMT. Recent studies point toan emerging role for MMPs in modulating aspects of immunity andinflammation during tumorigenesis. A variety of cytokines, cytokinereceptors, and chemokines have been found to undergo MMP-mediatedcleavage. In breast cancer, MMP-9 expression is upregulated intumor-associated stromal cells including neutrophils, macrophages, andlymphocytes and may play a role in tumor-associated inflammation.Several members of the MMP and ADAM family can regulate cellularproliferation by modulating the bioavailability of growth factors orcell-surface receptors. Ligands for several growth factor receptors areprocessed by MMP/ADAM family members as well. There are known clinicalbenefits of MMP inhibition in cancer management (for example Neovastat(AstraZeneca) is currently under evaluation in phase II renal cellcarcinoma). However, most MMP inhibitors are too toxic for use in theclinic and adverse effects of MMP inhibitors (e.g. musculoskeletaladverse effects) limit their use. Furthermore, there may be problemswith potent, broad spectrum, MMP inhibition. For example, there are somedata suggesting that tumour progression is inversely proportional toMMP-3. Accordingly, it is not known if MMP-3 sparing or MMP-3 inhibitingeffects are preferable. Recent developments in anti-cancer agentstargeting the matrix metalloproteinases have been reviewed (Li, et al.,Recent Patents on Anti-Cancer Drug Discovery 2010, 5: 109-141) and showthat MMP inhibitors are classified into three main pharmacologiccategories: Collagen peptidomimetics, non-peptidomimetics andtetracycline derivatives. Collagen peptidomimetics can be furthersubdivided into hydroxamates, carboxylates, aminocarboxylates,sulfhydryls, phosphoric acid derivatives. Most MMP inhibitors inclinical development are hydroxamate derivatives, e.g. batimastat andmarimastat, illomastat. The lead compounds have been largelyunsuccessful because of toxicity and or lack of efficacy. For example,Batimastat can only be administered intraperitoneally and intrapleurallyand further development has been suspended. In the case of Marimastat,no benefit over placebo was seen in patients with breast and lungcancer. Severe musculoskeletal pain occurred in 18% of patients andquality of life worsened with marimastat therapy. Development of thisdrug has also been discontinued. Several members of thenon-peptidomimetics class of compounds are undergoing evaluation inPhase III studies in cancer patients. However, the majority are nolonger in development because of an adverse efficacy/toxicity profile(including AG3340/Prinomastat (Agouron), BMS-275291(Bristol-Myers-Squibb), CGS27023A/MMI270 (Novartis),Bay12-9566/Tanomastat (Bayer Inc). Neovastat/AE-941 (Aetherna Zentaris)has MMP-2, MMP-9 and VEGF inhibitory properties and is being evaluatedas a potential treatment of renal carcinoma and Phase II clinical trialsare underway. Some tetracycline derivatives, such as doxycycline andCOL-3 have been evaluated in preclinical cancer models and G31 haveentered early clinical trials in patients. Doxycycline has been shown tosubstantially reduce the tumor burden from breast cancer metastasis innude mice. It exerts diverse inhibitor effects on MMP production andactivity, inhibits tumor cell proliferation. However, it accumulates athigh concentrations in bone, and can therefore be used for the treatmentof bone metastasis. Inhibition of mitochondrial protein synthesis bydoxycycline has significant anti-tumor effects in several tumor systems.Continuous doxycycline treatment combined with intermittentadministration of adriamycin or 1-beta-D-arabinofuranosyl cytosine onthe growth of rat leukemia resulted in the delay of tumor relapse.Treatment with zoledronic acid in combination with doxycycline may bevery beneficial for breast cancer patients at risk for osteolytic bonemetastasis, according to the fact that administration of a combinationof zoledronic acid and doxycycline resulted in a 74% decrease in totaltumor burden compared to untreated mice. In addition, doxycyclinesignificantly enhances the tumor regression activity ofcyclophosphamide, a widely used chemotherapeutic drug in neoplasias, onxenograft mice model bearing MCF-7 cells, suggesting that suchcombination chemotherapeutic regimen may lead to additional improvementsin treatment of breast cancer. In vivo, the inhibitory effects ofdoxycycline on breast cancer tumor matastasis formation was potentiatedby the addition of batimastat, confirming that targeting MMPs throughmultiple distinct pathways may improve treatment efficacy. However, in aPhase I evaluation of cancer patients, oral doses of 400 mg administeredtwice a day resulted in dose-limiting toxicity that consisted offatigue, confusion, nausea, and vomiting. At the maximum tolerated doseof 300 mg twice a day, mean through plasma concentrations werecomparable to those associated with antiangiogenic effect in vivo.

Nitric oxide is a gaseous molecule that is unsuitable for oraladministration. However, there are several pharmacologically relevantnitric oxide-donor groups than are known to release nitric oxide inresponse to conditions found in the human body after administration.Exemplary nitric-donor groups are described in “Nitric Oxide Donors: ForPharmaceutical and Biological Applications”; Peng George Wang, TingweiBill Cai, Naoyuki Taniguchi, Wiley (2005), the contents of which areincorporated herein by reference. The effects of nitric oxide on MMPsare complex. Nitric oxide has been reported to possess inhibitoryeffects on MMP-9 by destabilization of MMP-9 RNA and through effects onMMP-9 activating cytokines, secondary messengers and transcriptionfactors (AP-1). In contrast higher concentrations of nitric oxide havebeen shown to cause MMP activation through S-nitrosylation of aninhibitory cysteine on the prodomain.

Abbreviations: ALVDD=Asymptomatic left ventricular diastolicdysfunction, AUC=Area under the curve, cGMP=Cyclic guanosinemonophosphate, DMSO=Dimethyl supfoxide, DNA=Deoxyribonucleic acid,ECM=Extracellular matrix, FCS=Fetal calf serum, HCF=Human ventricularcardiac fibroblasts, HF=Heart failure, HFpEF=Heart failure withpreserved ejection fraction, IF=Interferon, iNOS=Inducible nitric oxidesynthase, IQR=Interquartile range, MCP=Monocyte chemotactic protein,MMP=Matrix metalloproteinase, MRI=Magnetic resonance imaging,mRNA=messenger ribonucleic acid, NHP=Non-human primate, NO=Nitric oxide,PBMC=Peripheral blood mononuclear cells, PCR=Polymerase chain reaction,RAAS=Renin-angiotensin-aldosterone system, RNA=Ribonucleic acid,SEM=Standard error of the mean, TIMP=Tissue inhibitor of matrixmetalloproteinase, TNFα=Tumor necrosis factor alpha.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda combination of a:

a first tetracycline (TC) component; and

a second component capable of releasing nitric oxide (NO) or a nitratecapable of mimicking NO effects in vivo (NO mimetic)

Nitric oxide (NO) is a gaseous molecule that is unsuitable for oraladministration. There are several pharmacologically relevant groups thanare known to liberate nitric oxide in response to conditions found inthe human body after administration or known to mimic nitric oxide'sactions, for example, by stimulating cGMP production. The nitratecapable of mimicking NO effects in vivo do not necessarily release NO,but may mimic NO effects in the body with actual release of NO from thesecond component. For example, one simple NO mimicry effect in vivo isthe activation of soluble guanyl cyclase (sGC) leading to elevated cGMP.The inventors have found that combinations of tetracyclines and nitricoxide donors or nitric oxide mimetics such as organic nitrates havefavourable effects on MMP expression in vivo. It has been found thatnitric oxide release or mimicry through sGC activation when combinedwith tetracyclines can achieve clinically relevant improvements in MMPmodulation efficacy and selectivity relative to tetracyclines bythemselves. The combinations of the invention advantageously act aseffective MMP modulators (with inhibitory effects on MMP-2 and MMP-9, inparticular), and/or modulators of inflammation mediators. Therefore, thevarious combinations of the invention find utility in medicalapplications where MMPs and/or inflammation is implicated. Included are,for example, myocardial interstitial disease, cardiac fibrosis, heartfailure such as heart failure with diastolic heart failure (DHF), heartfailure with preserved ejection fraction (HFpEF), congestive heartfailure (CHF), asymptomatic left ventricular diastolic dysfunction(ALVDD), coronary atherosclerosis (inflammation effects), cancers(through effects on tumor angiogenesis, tumor growth and metastasis) anddiabetes (inflammation effects). In addition, the combinations of theinvention are useful in other inflammatory diseases or diseasesassociated with inflammation, including but not limited to, inflammatorybowel disease, chronic prostatitis, infections, pulmonary inflammation,osteomyelitis, renal disease, gout, arthritis and shock.

The term “combination” is intended to cover related aspects of theinvention wherein (i) the first and second components are associatedtogether through a chemical interaction, such as a covalent bond or anelectrostatic interaction or a linker group to form a compoundcomprising both tetracycline and component capable of releasing nitricoxide (NO) or mimicking its effects (NO mimetic), or (ii) thetetracycline component and the component capable of releasing nitricoxide (NO) or NO mimetic are provided in the form of an admixture ofboth components, for example, in a single dosage unit; or (iii) thetetracycline component and the component capable of releasing nitricoxide (NO) or NO mimetic, are provided in the form of two or morecompositions, for example, separate dosage units, suitable foradministration to a patient to provide the desired therapeutic effect.

By “capable of releasing nitric oxide”, it is meant the dissociation orrelease in vivo of a nitric oxide molecule from the compound of theinvention, such that the nitric oxide component is no longer associatedwith, or linked to the tetracycline component or it is meant that thecomponent can mimic NO's effects in vivo such as through activation ofsGC.

By “nitric oxide releasing group” or NO mimetic, it is meant apolyatomic substance comprising at least one group capable of releasingnitric oxide or mimicking its effects. Such as group may be a nitrateester of an alkyl alcohol (organic nitrate). Alternatively the nitricoxide donor or mimetic group may be the conjugate base of nitric acid(nitrate ion). As explained above, second component molecules comprisingother nitric oxide mimetic or donor groups are also possible and includenitrate ester, diazeniumdiolates, N-diazen-1-ium-1,2-diolate (NONOate),S-nitrosothiols, furoxan or L-arginine which is a substrate for nitricoxide synthase. Clinically used nitric oxide mimetic or donor groupsthat may suitably be used in the combinations of the invention includeisosorbide dinitrate, isosorbide 2- and 5-mononitrate, erithrityltetranitrate, penterithrityl tetranitrate, nicorandil, sinitrodil,glyceryl trinitrate. Preferred “nitric oxide releasing group” or NOmimetics are is arginine, a metal nitrate salt or an aza-C₁ to C₅ alkyl,aza-C₁ to C₅ alkenyl, or aza-C₁ to C₅ alkynyl groups or a hetrocyclicamine group, which is substituted with at least one NO releasing group.Preferably the “nitric oxide releasing group” or NO mimetic is nitrate.In this embodiment, the preferred nitrate esters are selected fromH₂N-Et-ONO₂, HN-(Et-ONO₂)₂, MeNH-Et-ONO₂, Me₂N-Et-ONO₂, H₂N-pentyl-ONO₂or H₂N-cyclopentyl-ONO₂,

By “associated with” is meant that the tetracycline and the group ormolecule capable of releasing nitric oxide (NO) are associated, orlinked together by at least one chemical interaction. For example, anionic or electrostatic interaction, a covalent or a donor bondinteraction. This functional group may be involved in at least one ofthese types of chemical interaction with the tetracycline componenteither directly through covalent bonding or through electrostaticinteractions, or indirectly through a linker component, such as a linkergroup or molecule, for example, a chemical functional group or molecule.

In a first aspect, the combination of the invention concerns a compoundcomprising the first and second components. In a second aspect, thecombination of the invention concerns an admixture of at least one ofthe first and at least one of second components. In a third aspect, thecombination of the invention concerns two or more separate compositionsof at least one of first and at least one of the second components foradministration. Accordingly, the second component may be mixed with,administered with, bonded or linked with the first tetracyclinecomponent as described above for the purposes of the combinations of thepresent invention.

Preferably, the combination is a compound in which the first and secondcomponents are associated together through a chemical interaction, suchas a covalent bond or an electrostatic interaction or a linker group toform a compound comprising both tetracycline and component capable ofreleasing nitric oxide (NO) or mimicking its effects (NO mimetic).

In this embodiment, the components are associated together through achemical interaction, such as a covalent bond or an electrostaticinteraction or a linker group to form a compound comprising bothtetracycline and component capable of releasing nitric oxide (NO) ormimicking its effects (NO mimetic). Suitably, the linker is a methylene(—CH₂—), or methylene substituted with a methyl, ethyl or propyl group(—CHMe-, —CHEt- or CHPr—).

Accordingly, in the first aspect, the compound of the inventioncomprises a first tetracycline component having general formula:

in which

-   R₁ is —H or —OH;-   R₂ is —H, —OH or -Me;-   R₃ is —H, or —NMe₂;-   R₄ is —H, —OH or -Me; and-   R₅ is —H or —OH. Preferably, when R₂ is —H or Me, R₄ is —OH.    Preferably, the tetracycline may be selected from the group    consisting of: tetracycline, minocycline, doxycycline and    oxytetracycline. The structures of these tetracyclines are:

In a particularly preferred embodiment, the tetracycline may bedoxycycline, doxycycline hyclate or doxycycline hydrochloride.

In the first aspect, in which the combination of the invention concernsa compound, the second component is designated herein as the “associatedmolecule” or as the “second component”. The second component is eithercapable of mimicking NO's effects in vivo, capable of releasing nitricoxide spontaneously, or is capable of releasing nitric oxide (NO)through metabolism to form nitric oxide or at least one of its redoxcongeners. Preferred redox congeners of nitric oxide include any reducedform of nitric oxide (NO). They may be selected from nitroxyl anion(NO⁻), NO radical (NO.) and nitrosonium cation (NO⁺═N≡O⁺). The skilledperson will appreciate that the form of nitric oxide redox congenerproduced will depend on various enzymatic or non-enzymatic metabolicpathways involved in any particular nitric acid metabolism of thecompounds of the invention. Preferably, in this embodiment, the secondcomponent comprises at least one functional group comprising N(O)_(n)which is associated with the tetracycline; wherein n is an integerselected from 1-3. Suitably, the at least one functional groupcomprising N(O)_(n) is capable of releasing nitric oxide (NO) or actingas an NO mimetic. Preferably n is 3. Suitably, the second componentforms at least one chemical bond with the tetracycline component of thecompound of the invention. Preferable the chemical bond may be acovalent, a polar covalent bond or a donor (coordinate) bond between thefirst and second components. Preferably, the bond is a covalent bond.Alternatively, the second component may be associated, or linked,directly with the first tetracycline component through an electrostaticor ionic interaction.

Further alternatively, the second component may be associated, orlinked, with the first tetracycline component through a linker group ormolecule, which are described below in more detail.

Accordingly, in the first aspect, wherein the combination of theinvention concerns a compound, the compound comprises:

a first tetracycline (TC) component; and

a second component capable of releasing nitric oxide (NO) or mimickingnitric oxide (NO);

wherein the second component is ionically or covalently bonded to thefirst component, or is linked thereto, by means of a linker atom ormolecule. Preferably, the second component is covalently bonded to thefirst component. More preferably still, the second component is linkedto the first component by a linker atom or molecule. Preferably, thesecond component comprises at least one functional group havingN(O)_(n), wherein n is 3. In this embodiment, the at least onefunctional group comprises a nitrate anion (NO₃ ⁻). Alternatively, thefunctional group comprises a nitrate group (—ONO₂). Nitrate compoundsare particularly preferred because of their clinical use, stability andlipophilicity.

In a preferred embodiment of the first aspect of the invention, thecompound is a tetracycline nitrate ester, in which nitrate is directlybonded to the tetracycline component. In this embodiment, the compoundof the invention takes the form tetracycline (TC)—NO₂, where no linkergroup or molecule is required. For example, the compound of theinvention is doxycycline nitrate, structure shown below. The skilledperson will appreciate where the nitrate group can be directly bonded tothe tetracycline in this embodiment.

Alternatively, the linker group may be a compound forming a Mannich baseattachment to the tetracycline (TC-M-ONO₂). Where a linker group isused, more than one nitrate can be appended onto the linker, forexample, (TC-M-(ONO₂)₂). Finally, the second component molecule maysimply be the counter anion (ONO₂), wherein an ionic interaction betweena cationic form of the tetracycline (TC⁺) and the nitrate anion providesthe basis for the association between the TC and second component of thecompound of the invention. The Mannich base is formed by reaction of theamide group of the first tetracycline component with formaldehyde or analdehyde to form an imine, which is subsequently reacted with an amineforming a Mannich base derivative. Alternatively the TC can be reactedwith an immine formed by reacting an aldehyde with amine. In thisembodiment, the compound of the invention takes the form tetracycline(TC)-M-ONO₂, where M represents the Mannich base attachment or thelinker group created by the Mannich base attachment (for example,methylene (—CH₂—), or methylene substituted with a methyl, ethyl orpropyl group (—CHMe-, —CHEt- or CHPr—), etc, depending on the aldehydeused in the Mannich base reaction), which leads to insertion of thelinker group. The Mannich base attachment is to the primary amide of thetetracycline of the invention. The skilled person will appreciate thatthen, for example, an aldehyde such as paraformaldehyde is used to formthe Mannich base linkage, the reaction inserts a methylene group betweenthe first and second components of the compound of the invention. Thismethylene group then serves as a linker associating the first and secondcomponents together by covalent bonding. Different types of methylenelinkers with different substitutions may be used by selection ofappropriate aldehyde.

Accordingly, in a second embodiment of the first aspect, wherein thecombination of the invention concerns a compound, the second componentcomprises:

(i) a short chain aza-alkyl (C₁ to C₅), aza-alkenyl (C₁ to C₅), oraza-alkynyl (C₁ to C₅) group, which can be linear, branched, or cyclic;and

(ii) at least one nitric oxide donor group or NO mimetic selected from anitrate ester, diazeniumdiolates a N-diazen-1-ium-1,2-diolate (NONOate),S-nitrosothiols, furoxan or a molecule capable of releasing nitric oxide(NO), such as arginine or similar nitric oxide releasing moiety ormimetic.

In this embodiment, the second component may be bonded or linked to thefirst tetracycline component through its primary amide by a covalentbond giving a compound with general structure:

in which R is one of the aza-alkyl (C₁ to C₅), aza-alkenyl (C₁ to C₅),or aza-alkynyl (C₁ to C₅) groups or fragments described above, Tc is thefirst tetracycline component, and L is a methylene linker, which can beunsubstituted (—CH₂—) or substituted with a methyl, ethyl or propylgroup (—CHMe-, —CHEt- or CHPr—). The substitution on the methylenelinker depends on the aldehyde used in the Mannich base reaction.Examples of the second compound include nitrated aza-alkyl, nitratedaza-alkenyl, or nitrated aza-alkynyl groups

In a second embodiment of the first aspect of the invention, the secondcomponent comprises:

(i) a short chain aza-alkyl (C₁ to C₅), aza-alkenyl (C₁ to C₅), oraza-alkynyl (C₁ to C₅) group or fragment, which can be linear, branched,or cyclic, and which can be substituted or unsubstituted; and

(ii) at least one nitric oxide donor or mimetic group or at least onegroup comprising N(O)_(n), wherein n is an integer selected from 1-3, asdefined above, hereinafter referred to as a ¢nitric-oxide donor group”.

Examples of the second component include aza-(C₁ to C₅)alkyl, aza-(C₁ toC₅)alkenyl aza-(C₁ to C₅)alkynyl group or fragment which is substitutedwith at least one nitrate. Preferred examples of the second componentinclude nitrated aza-alkyl, wherein the second component is a nitratedC₁ to C₅ alkyl amine, more preferably, a nitrated C₁ to C₂ alkyl amine.Preferably, the nitrated aza-alkenyl is a nitrated C₁ to C₅ alkenylamine, more preferably a nitrated C₁ to C₂ alkenyl amine. Preferably,the nitrated aza-alkynyl group is a nitrated C₁ to C₅ alkynl amine, morepreferably a nitrated C₁ to C₂ alkynl amine. Preferred examples of thesecond component comprises a short chain aza-alkyl (alkyl amine)molecule H₂N—R, in which R is a C₁-C₅ alkyl group, more preferably aC₁-C₂ alkyl group. Preferably, the nitric oxide donor or mimetic groupis —ONO₂. Examples of the second component are be H₂N—R—N(O)_(n), inwhich R is a C₁-C₅ alkyl, alkenyl or alkenyl more preferably a C₁-C₂alkyl alkyl, alkenyl or alkenyl. The nitric oxide donor or mimetic groupin this example is preferably —ONO₂.

In this embodiment, the second component is linked to the firsttetracycline component by a Mannich base attachment through a linker Lto the amide group of the first tetracycline component, giving acompound with general structure:

in which R is a aza-alkyl (C₁ to C₅), aza-alkenyl (C₁ to C₅), oraza-alkynyl (C₁ to C₅) group or fragment, Tc is the first tetracyclinecomponent, and L is a methylene linker, which can be unsubstitutedmethylene (—CH₂—) or substituted with a methyl, ethyl or propyl group(—CHMe-, —CHEt- or CHPr—).

In a third embodiment of the first aspect of the invention, the secondcomponent may be linked to the first tetracycline component by a Mannichbase attachment through a linker group L to the amide group of the firsttetracycline component, forming a compound with general structure:

wherein R is an aza-alkyl (C₁ to C₅), aza-alkenyl (C₁ to C₅), oraza-alkynyl (C₁ to C₅) group or fragment, Tc is the first tetracyclinecomponent, and L is a methylene linker. The methylene linker may beunsubstituted (—CH₂—) or substituted with a methyl, ethyl or propylgroup (—CHMe-, —CHEt- or CHPr—). The substitution depends on thealdehyde used in the Mannich base reaction.

In a fourth embodiment of the first aspect of the invention, the secondcomponent comprises:

(i) an aza-ethyl molecule (ethyl amine, EtNH₂); and

(ii) at least one nitrate (—ONO₂) group.

Typical examples of second component molecules are H₂N-Et-ONO₂.

In this embodiment, the second component is linked to the firsttetracycline component by a Mannich base attachment through a linker Lto the amide group of the first tetracycline component, forming acompound with general structure:

in which Tc is the first tetracycline component, and L is a methylenelinker. The methylene linker may be unsubstituted (—CH₂—) or substitutedwith a methyl, ethyl or propyl group (—CHMe-, —CHEt- or CHPr—).

In a preferred fifth embodiment of the first aspect, the secondcomponent comprises:

(i) an aza-dimethylethyl molecule (dimethylethyl amine, Me₂NEt); and

(ii) at least one nitrate (—ONO₂) group

Typical examples of second component molecules in this example take theform Me₂N-Et-ONO₂.

In this embodiment, the second component is linked to the firsttetracycline component by a Mannich base attachment through a linker Lto the amide group of the first tetracycline component, forming acompound with general structure:

in which Tc is the first tetracycline component, and L is a methylenelinker, which can be unsubstituted (—CH₂—) or substituted with a methyl,ethyl or propyl group (—CHMe-, —CHEt- or CHPr—).

In a preferred sixth embodiment of the first aspect, the secondcomponent comprises:

(i) an aza-diethyl molecule (diethyl amine, EtNHEt); and

(ii) at least one nitric oxide releasing or NO mimetic.

Typical examples of second components in this embodiment take the formEtHN-Et-N(O)_(n), wherein n=1 to 3. Preferably n=3.

In this embodiment, the second component is linked to the firsttetracycline component by a Mannich base attachment through a linker Lto the amide group of the first tetracycline component, forming acompound with general structure:

in which Tc is the first tetracycline component, and L is a methylenelinker, which can be unsubstituted (—CH₂—) or substituted with a methyl,ethyl or propyl group (—CHMe-, —CHEt- or CHPr—). The linker substituentdepends on the aldehyde used in the Mannich base reaction.

In a preferred seventh embodiment of the first aspect, the secondcomponent comprises an aza-diethyl molecule (diethyl amine, EtNHEt ordiethyl methylamine EtNMeEt) and two nitrate groups, wherein one nitrategroup (—ONO₂) is attached to each ethyl group. Typical examples ofsecond components in this example take the form HN-(Et-ONO₂)₂.Accordingly, in such embodiments, the second component capable ofreleasing nitric oxide (NO) having the at least one nitric oxide donorgroup is N,N-di-ethylnitrate amine.

In this embodiment, the second component is linked to the firsttetracycline component by a Mannich base attachment through a linker Lto the amide group of the first tetracycline component, forming acompound with general structure:

in which Tc is the first tetracycline component, and L is a methylenelinker, which can be unsubstituted (—CH₂—) or substituted with a methyl,ethyl or propyl group (—CHMe-, —CHEt- or CHPr—). The substituent dependson the aldehyde used in the Mannich base reaction.

In a preferred eight embodiment of the first aspect, the secondcomponent comprises:

(i) an aza-pentyl molecule (pentyl amine, pentyl-NH₂) or anaza-cyclo-pentyl molecule; and

(ii) at least one at least one nitrate (—ONO₂) group.

Typical examples of second components in this example take the formH₂N-pentyl-ONO₂ or H₂N-cyclopentyl-ONO₂. The second component iscovalently bonded or linked to the first tetracycline component throughthe Mannich base attachment described above.

In a preferred ninth embodiment of the first aspect of the invention,the second component comprises a heterocyclic amine, which can besubstituted or unsubstituted. Suitably, the nitric oxide releasing groupor the NO mimetic can be linked to the heterocyclic amine at the 2, 3,or 4 positions. Preferably, the heterocyclic amine may be selected frompiperidine, piperazine or pyrrolidine. The heterocyclic amine may besubstituted with a direct —ONO₂ group or a linker-ONO₂, wherein thelinker is a C₁-C₅ alkyl group or more preferably a C₁-C₂ alkyl group.

In a preferred tenth embodiment of the first aspect of the invention,the second component comprises:

(i) a piperidine molecule; and

(ii) at least one at least one nitrate (—ONO₂) group.

Examples of the second component include:

In this embodiment, the second component is linked to the firsttetracycline component by a Mannich base attachment through a linker Lto the amide group of the first tetracycline component, forming acompound with general structure:

in which, Tc is the first tetracycline component, and L is a methylenelinker, which can be unsubstituted (—CH₂—) or substituted with a methyl,ethyl or propyl group (—CHMe-, —CHEt- or CHPr—).

In a preferred eleventh embodiment, the second component comprises acombination of:

(i) a piperidine molecule; and

(ii) at least one at least one alkyl-nitrate (R—ONO₂) group, in which Ris a C₁-C₅ alkylene group. More preferably R is a C₁ to C₂ alkylenegroup. Most preferably, the R group is a methylene (—CH₂—) group. Theskilled person will appreciate that the alkyl nitrate group may besubstituted at the 2, 3 or 4 position of the piperidine ring.Preferably, the at least one alkyl nitrate group (-alkyl-ONO₂) is at the3 position of the ring.

In this embodiment, the second component is linked to the firsttetracycline component by a Mannich base attachment through a linker Lto the amide group of the first tetracycline component, forming acompound with general structure:

in which Tc is the first tetracycline component, L is a methylenelinker, which can be unsubstituted (—CH₂—) or substituted with a methyl,ethyl or propyl group (—CHMe-, —CHEt- or CHPr—), and R is a C₁-C₅alkylene group. More preferably R is C₁ to C₂ alkylene group. Mostpreferably, R is methylene.

In a preferred twelfth embodiment, the second component comprises:

(i) a piperidine molecule; and

(ii) at least one at least one —CH₂ONO₂ group

Suitably, the methyl-nitrate groups can be linked at the 2, 3, or 4 ringpositions. Position 3 is the most preferred position. Typical examplesof second component molecules in this example include:

In this embodiment, the second component is linked to the firsttetracycline component by a Mannich base attachment through a linker Lto the amide group of the first tetracycline component, forming compoundwith general structure:

in which, Tc is the first tetracycline component, L is a methylenelinker, which can be unsubstituted (—CH₂—) or substituted with a methyl,ethyl or propyl group (—CHMe-, —CHEt- or CHPr—).

In a preferred thirteenth embodiment, the second component is a nitrateanion. The nitrate anion is involved in at least one ionic orelectrostatic interaction with the tetracycline component. In an exampleof this embodiment, the second component may be metal nitrate salt.Preferably, the nitrate salt is silver nitrate. Silver nitrate forms anitrate ionic salt with the tetracycline (TC⁺NO₃ ⁻). Typically, anitrate salt can be formed by reaction of the TC hydrochloride withAgNO₃, for example.

The preferred compounds of the invention may be selected from:

wherein linker L is a methylene (—CH₂—), or methylene substituted with amethyl, ethyl or propyl group (—CHMe-, —CHEt- or CHPr—), R is a C₁-C₅alkylene group.

In a preferred fourteenth embodiment of the first aspect of theinvention, the second component may comprise arginine. Preferably, thesecond component molecule is L-arginine. L-arginine is a substrate fornitric oxide synthase (NOS) and its metabolites include nitric oxide(NO). Suitably, the L-arginine may be linked to the first tetracyclinecomponent through a linker group or molecule as described above.

In a preferred fifteenth embodiment of the first aspect of theinvention, the compound may be selected from the group consisting of:

-   6-deoxy-5-oxytetracycline nitrate salt;-   doxycycline-5-nitrate

-   doxycycline-12a-nitrate;-   minocycline-12a-nitrate;-   amido-N-[3-methylnitratepiperidinomethy]-α-6-deoxy-5-oxytetracycline;-   amido-N—[N,N-diethylnitrate-aminomethyl]-α-6-deoxy-5-oxytetracycline    (amido-N—[bis-(β-nitrooxyethyl)aminomethyl]-α-6-deoxy-5-oxytetracycline)

-   amido-N-[(3-nitrooxyethyl)aminomethyl]-α-6-deoxy-5-oxytetracycline

-   amido-N-[3-(nitrooxymethyl)piperidinomethyl]-α-6-deoxy-5-oxytetracycline

-   amido-N-[3-(nitrooxymethyl)piperidinomethyl]-α-6-deoxy-5-oxytetracycline

-   amido-N-[4-(nitrooxymethyl)piperidinomethyl]-α-6-deoxy-5-oxytetracycline

-   amido-N-[4-nitrooxypiperidinomethyl]-α-6-deoxy-5-oxytetracycline

-   amido-N-[4-nitrooxypiperidinomethyl]-tetracycline

-   amido-N—[bis-(β-nitrooxyethyl)methylaminomethyl]-α-6-deoxy-5-oxytetracycline

-   amido-N—[bis-(β-nitrooxyethyl)methylaminomethyl]-α-6-deoxy-5-oxytetracycline

-   amido-N—[bis-(β-nitrooxyethyl)ethylaminomethyl]-tetracycline

-   amido-N-[(β-nitrooxyethyl)aminomethyl]-tetracycline

-   amido-N-[4-(nitrooxymethyl)piperidinomethyl]-tetracycline

-   amido-N-[3-(nitrooxymethyl)piperidinomethyl]-tetracycline

In the second aspect of the invention, the combination of the inventionmay be provided as an admixture of the first and second components ofthe invention. An admixture means a mixture the components where theyare not chemically bonded or associated together on a molecular level.Preferably, in this aspect, at least one of the first tetracyclinecomponents described above may be mixed with at least one of the secondcomponents examples described above to form an admixture.

In the third aspect of the invention, the combination of the inventionmay be provided in the form of two or more separate compositions of atleast one tetracycline and at least one second component capable ofreleasing NO or otherwise mimicking the effect of NO in vivo, foradministration to a patient to provide the desired therapeutic effectachieved by the admixtures or compound of the invention.

Examples of the second components of the invention include aza-alkyl,aza-alkenyl, or aza-alkynyl groups which are substituted with at leastone NO releasing group, as defined above. Preferably, the NO releasinggroup is a nitrate group. Preferably, the nitrated aza-alkyl, comprisesa C₁ to C₅ alkyl, more preferably, a C₁ to C₂ alkyl group. Preferably,the nitrated aza-alkenyl comprises a C₁ to C₅ alkenyl, more preferably aC₁ to C₂ alkenyl group. Preferably, the nitrated aza-alkynyl comprises aC₁ to C₅ alkynl, more preferably a C₁ to C₂ alkynl group. Examples ofthe second component include H₂N—R—N(O)_(n), in which R is a C₁-C₅alkyl, alkenyl or alkenyl, more preferably a C₁-C₂ alkyl alkyl, alkenylor alkenyl group. The nitric oxide donor group in this example ispreferably —ONO₂. Typical examples of second component molecules areH₂N-Et-ONO₂, HN-(Et-ONO₂)₂, MeNH-Et-ONO₂, Me₂N-Et-ONO₂, H₂N-pentyl-ONO₂or H₂N-cyclopentyl-ONO₂. Further examples of second components include:

Suitably, the second component may be metal nitrate salt. Preferably,the nitrate salt is silver nitrate. Alternatively, the second componentmolecule may be L-arginine. Clinically used nitric oxide mimetic ordonor groups which may be used as second components within the variousaspect of the invention and include isosorbide dinitrate, isosorbide 2-and 5-mononitrate, erithrityl tetranitrate, penterithrityl tetranitrate,nicorandil, sinitrodil, glyceryl trinitrate. These clinically usednitrates are particularly preferred in the admixtures aspect of theinvention.

According to a fourth aspect of the present invention, there is provideda method of preparing an admixture comprising the step of:

(i) mixing together a first tetracycline component, and

(ii) a second component capable of releasing nitric oxide (NO) orcapable of otherwise mincking NO in vivo.

According to a fifth aspect of the present invention, there is provideda method of preparing a compound of the invention, the method comprisingthe step of:

(i) reacting together a first tetracycline component, and

(ii) a second component capable of releasing nitric oxide (NO) or actingas an NO mimetic,

such that the second component becomes ionically or covalently bonded tothe first component, or linked thereto, by means of a linker atom ormolecule. In other words, the first tetracycline component becomesassociated or linked with the molecule that is capable of releasingnitric oxide (NO) or mimicking its effects.

Preferably, the method comprises reacting a second component compoundhaving at least one functional group comprising N(O)_(n), wherein n isfrom 1 to 3, with a first tetracycline component, such that thetetracycline component becomes associated or linked with the compoundhaving at least one functional group comprising N(O)_(n). It will beappreciated that the second component is capable of releasing nitricoxide (NO).

The skilled person will appreciate that the second component that iscapable of releasing nitric oxide (NO) may be involved in at least onetype of chemical interaction with the first tetracycline componenteither directly through covalent bonding or through electrostaticinteractions, or indirectly through a linker, such as a chemicalfunctional group or molecule.

In a preferred embodiment, the second component reacts with the firsttetracycline component to form a Mannich base link with the tetracyclineprimary amide. It will be appreciated that this reaction occurs underconditions allowing Mannich base formation. Accordingly, in preferredembodiment, the reaction of the first and second components of thecompound of the invention occurs in the presence of an aldehyde.Preferably, the aldehyde is formaldehyde. More preferably still, thealdehyde is paraformaldehyde, which, in the Mannich base attachmentsresults in insertion of a methylene group between the first and secondcomponents.

In a preferred embodiment of the method of preparing the compound of theinvention, the second component molecule is provided in solution.Suitably, the second component is provided in solution with an alcohol.Preferably, the second component is provided in a secondary alcohol, forexample, isopropyl alcohol.

The second component is provided in solution with a secondary alcohol byheating to a temperature of 65-85° C., preferably 75° C. The secondcomponent provided in solution with a secondary alcohol is then reactedwith the first component at a temperature of 30-50° C., preferably 40°C.

Suitably, the first tetracycline component is provided in solution withan alcohol, an ether or a nitrile. Preferably, the alcohol may beselected from methanol, isopropyl alcohol, or a mixture thereof.Preferably, the ether is a polar ether, for example, tetrahydrofuran(THF). Preferably, the nitrile is acetonitrile.

It will be appreciated that the first tetracycline (TC) component andthe second component of the compound of the invention can be any of thetetracylines or second component molecules described herein. However, ina preferred embodiment, the tetracycline component is doxycycline. Thepreferred second component molecule is N,N-di-ethylnitrate amine.

The solution of the tetracycline component and the solution of thesecond component are mixed to start the reaction. Preferably, themixture of the tetracycline and the second component proceeds underconstant stirring. Suitably, the reaction is conducted at a temperatureof 20-50° C. Preferably, the reaction is conducted at a temperature of20° C. Alternatively, the reaction is conducted at a temperature of 40°C. The reactants may be stir to facilitate reaction for from about 0.5to about 18 hours. Preferable, the reaction is conducted for at least0.5 hour. Alternatively, the reaction is conducted for at least 2 hours.Further alternatively, the reaction is conducted for at least 16 hours.The skilled person will appreciate the time necessary for completion ofreaction will depend on the nature of the specific tetracyclinecomponent, the second component, their solubilities in the solvents ofchoice.

In a preferred embodiment, the method of preparing a compound accordingto the first aspect of the present invention comprises the step ofreacting a nitrate-containing group with tetracycline, optionally in thepresence of an aldehyde forming a Mannich base with the tetracyclineprimary amide.

Preferably, the aldehyde is formaldehyde. Further preferably, thealdehyde is paraformaldehyde.

Alternatively, the at least one nitrate-containing group comprises ametal nitrate. Optionally, the at least one nitrate-containing groupcomprises silver nitrate.

According to a sixth aspect of the present invention, there is provideda pharmaceutical composition comprising a combination of a:

a first tetracycline (TC) component; and

a second component capable of releasing nitric oxide (NO);

wherein the second component is combined with the first tetracyclinecomponent.

The term “combined” is intended to cover embodiments wherein (i) thefirst and second components are associated together through aninteraction of the types described below to form a compound comprisingboth tetracycline and component capable of releasing nitric oxide (NO),and (ii) the tetracycline component and the component capable ofreleasing nitric oxide (NO) are provided in the form of an admixture ofboth components, for example, in a single dosage unit; and (iii) thetetracycline component and the component capable of releasing nitricoxide (NO) are provided in the form of two or more separate dosage unitsfor substantially simultaneously administration to a patient.

Accordingly, in a preferred embodiment, the pharmaceutical compositioncomprises an admixture of the first tetracycline (TC) component; and thesecond component capable of releasing nitric oxide (NO). In aparticularly preferred embodiment, the pharmaceutical compositioncomprises a compound of the invention.

According to a seventh aspect of the present invention, there isprovided a method of treating a disease or condition by administering atherapeutically effective amount of the combination of the invention,wherein the combination comprises:

a first tetracycline (TC) component; and

a second component capable of releasing nitric oxide (NO);

wherein the second component is combined with the first tetracyclinecomponent.

Suitably, the method of treating a disease or condition comprisesadministering a therapeutically effective amount of the combination ofthe invention to a patient in need thereof. The combination may beadministered by providing the patient with a therapeutically effectiveamount of the compounds described herein. Alternatively, the combinationmay be administered by providing the patient with a therapeuticallyeffective amount of an admixture of the first tetracycline component andthe second component capable of releasing nitric oxide (NO).Alternatively still, the combination may be administered by providingthe patient with a therapeutically effective amount of the first andsecond components by co-administering the tetracycline and the secondcomponent capable of releasing nitric oxide (NO), as part of a suitabledosage regimen.

Accordingly, the combinations, the compound or the pharmaceuticalcompositions of the invention may be used in the medical field. Moresuitably, the combinations or the compounds of the invention or thepharmaceutical compositions comprising the combination or the compoundof the invention can be used in the medical field. The combination, thecompound or the pharmaceutical composition of the invention may be usedas a medicament or may be used in the manufacture of a medicament forthe treatment of, alleviation of, and/or prevention of a disease. In aparticularly preferred embodiment, the combination, the compound or thepharmaceutical composition of the invention may be used in the treatmentor prevention of inflammatory and/or cardiovascular diseases selectedfrom the group consisting of: myocardial interstitial disease, cardiacfibrosis, heart failure such as heart failure with diastolic heartfailure (DHF), heart failure with preserved ejection fraction (HFpEF),congestive heart failure (CHF), asymptomatic left ventricular diastolicdysfunction (ALVDD), coronary atherosclerosis (inflammation effects),cancers (through effects on tumor angiogenesis, tumor growth andmetastasis) and diabetes (inflammation effects), inflammatory boweldisease, chronic prostatitis, infections, pulmonary inflammation,osteomyelitis, renal disease, gout, arthritis and shock.

With regard to the admixtures combination aspect of the invention.Admixture of doxycycline and nitrate A (diethanolamine dinitrate), inparticular, can be used to treat invasive bladder cancer, chronicprostatitis, acute pyelpnephritis, non-Hodgkins lymphoma, pulmonaryinfections and osteomyelitis through the effect on IL8. Whereas, anadmixture of doxycycline and nitrate A (Diethanolamine dinitrate), inparticular, can be used to treat inflammatory bowel disease througheffects on IL4. Furthermore, admixtures of doxycycline and nitrate A(Diethanolamine dinitrate) can be used to treat fever, anemia,cryopyrinopathies (hereditary periodic fever syndromes), gout andpseudogout, Septic shock (IL-1β). Alternatively, admixtures ofdoxycycline and nitrate B (Nitroxymethyl piperidine) can be used totreat fever, anemia, cryopyrinopathies (hereditary periodic feversyndromes), gout and pseudogout, Septic shock (IL-1β). However,admixtures of doxycycline and nitrate A (Diethanolamine dinitrate) arepreferred in treating these particular conditions.

With regard to use of the compounds of the invention, preferably, thedisease is a cardiovascular disease, such as heart failure. In aparticularly preferred embodiment, the combination, the compound or thepharmaceutical composition of the invention is used in treatment orprevention of heart failure caused by or associated with diastolicdysfunction.

In a preferred embodiment, the combinations, the compounds or thepharmaceutical compositions of the invention may be used in thetreatment of cancer. Suitably, the cancer may be at least one of thegroup consisting of: bone metastasis, breast cancer, pancreatic cancer,lung cancer, bladder cancer, colorectal cancer, ovarian cancer, prostatecancer, gallbladder cancer or cancerous brain tumors. Suitably, thecancer is breast or colorectal cancer. The combinations and compoundsdescribed herein may be used in conjunction with other drug actives ortherapeutic agents known to the skilled person. The other therapeuticagent can provide additive or synergistic value relative to theadministration of the combination or the compound of the inventionalone, and may be selected from lipid-lowering agents that reduce bloodlevels of cholesterol and trigylcerides, agents that normalize bloodpressure, agents, such as aspirin or platelet ADP receptor antatoginist(e.g., clopidogrel and ticlopidine), that prevent activation ofplatelets and decrease vascular inflammation, and pleotrophic agentssuch as peroxisome proliferator activated receptor (PPAR) agonists, withbroad-ranging metabolic effects that reduce inflammation, promoteinsulin sensitization, improve vascular function, and correct lipidabnormalities. Further advantages may arises from combination withanother therapeutic agent for cardiovascular disease. Examples of suchagents include, but are not limited to an anti-inflammatory agent, anantithrombotic agent, an anti-platelet agent, a fibrinolytic agent, alipid reducing agent, a direct thrombin inhibitor, a glycoproteinIIb/IIIa receptor inhibitor, an agent that binds to cellular adhesionmolecules and inhibits the ability of white blood cells to attach tosuch molecules, a calcium channel blocker, a beta-adrenergic receptorblocker, a cyclooxygenase-2 inhibitor, an angiotensin system inhibitor,and/or combinations thereof. Antiinflammatory agents includeimmunosuppressants, TNF inhibitors, corticosteroids, non-steroidalanti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs(DMARDS), and the like. Exemplary antiinflammatory agents include, forexample, prednisone; methylprenisoione (Medrol®), triamcinolone,methotrexate (Rheumatrex®, Trexall®), hydroxychloroquine (Plaquenil®),sulfasalzine (Azulfidine®), leflunomide (Arava®), etanercept (Enbrel®),infliximab (Remicade®), adalimumab (Humira®), rituximab (Rituxan®),abatacept (Orencia®), interleukin-1, anakinra (Kineret™), ibuprofen,ketoprofen, fenoprofen, naproxen, aspirin, acetominophen, indomethacin,sulindac, meloxicam, piroxicam, tenoxicam, lornoxicam, ketorolac,etodolac, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamicacid, diclofenac, oxaprozin, apazone, nimesulide, nabumetone, tenidap,etanercept, tolmetin, phenylbutazone, oxyphenbutazone, diflunisal,salsalate, olsalazine or sulfasalazine. The additional therapeutic maybe a chemotherapeutic drugs or anti-proliferative agent selected fromalkylating agents, antimetabolites, anthracyclines, plant alkaloids,topoisomerase inhibitors, or other antitumour agents, monoclonalantibodies, tyrosine kinase inhibitors, hormones. Exemplaryanti-proliferative agents include vinca alkaloids (e.g. vinblastine),the anthracyclines (e.g. adriamycin), the epipodophyllotoxins (e.g.etoposide), antibiotics (e.g. actinomycin D and gramicidin D),antimicrotubule drugs (e.g. colchicine), protein synthesis inhibitors(e.g. puromycin), toxic peptides (e.g. valinomycin), topoisomerase Iinhibitors (e.g. topotecan), DNA intercalators (e.g. ethidium bromide),anti-mitotics, vinca alkaloids (e.g. vinblastine, vincristine, vindesineand vinorelbine), epothilones (e.g. epothilone A, epothilone B anddiscodermolide), nocodazole, colchicine, colchicine derivatives,allocolchicine, Halichondrin B, dolstatin 10, maytansine, rhizoxin,thiocolchicine, trityl cysterin, estramustine, nocodazole,platinum-based agents (e.g. cisplatin, paraplatin, carboplatin, but notthe subject platinum-based chemotherapeutic agents as defined herein),camptothecin, 9-nitrocamptothecin (e.g. Orethecin, rubitecan),9-aminocamptothecin (IDEC-13′), exatecan (e.g. DX-8951f), lurtotecan(GI-147211 C), BAY 38-3441, the homocamptothecins such as diflomotecan(BN-80915) and BN-80927, topotecan (Hycamptin), NB-506, J107088,pyrazolo[1,5-a]indole derivatives, such as GS-5, lamellarin D,irinotecan (Camptosar, CPT-11), and antibodies, such as 1 D1 0, Hu1D10,1 D09C3, 1C7277, 305D3, rituximab, 4D5, Mab225, C225, Daclizumab(Zenapax), Antegren, CDP 870, CMB-401, MDX-33, MDX-220, MDX-477,CEA-CIDE, AHM, Vitaxin, 3622W94, Therex, 5G1.1, IDEC-131, HU-901,Mylotarg, Zamyl (SMART M195), MDX-210, Humicade, LymphoCIDE, ABX-EGF,17-1A, Epratuzumab, Cetuximab (Erbitux), Pertuzumab (Omnitarg, 2C4), R3,CDP860, Bevacizumab (Avastin), tositumomab (Bexxar), Ibritumomabtiuxetan (Zevalin), M195, 1D10, Hu1D10 (Remitogen, apolizumab),Danton/DN1924, an “HD” antibody such as HD4 or HD8, CAMPATH-1 andCAMPATH-1H or other variants, fragments, conjugates, derivatives andmodifications thereof, or other equivalent compositions with improved oroptimized properties.

For example, it is known in the art that doxycycline with zoledronicacid is useful in breast cancer treatment. Adriamycine and1-beta-D-arabinofuranosykl cytoside combinations are useful in delay oftumor relapse. Combination with cyclophosphamide may also be useful inchemotherapy.

In a eighth aspect of the invention, the combinations or the compoundsdescribed herein may be used in a screening method to identify furthercompounds having benefits in the disease states mentioned above. In aninth aspect, the combinations or the compounds described herein may beused in determining the suitable of the combination and/or the compoundsof the invention for the treatment the disease states mentioned herein.

Further Definitions For the purposes of this specification, in the caseof a polyatomic molecule represented by text, a single bond extendingbetween any two atoms is represented by a solid dashed line (—), adouble bond extending between any two atoms is represented by a doublesolid dashed line (═), and a triple bond extending between any two atomsis represented by a triple solid dashed line (≡), unless otherwisestated. By “short chain” is meant a polyatomic molecule comprising atleast one carbon atom. Optionally, the polyatomic molecule comprises 1-6carbon atoms. Further optionally, the polyatomic molecule comprises 1-3carbon atoms. By the term “linear” is meant a molecule comprising atleast two atoms, any of which can be the same or different, wherein eachatom of the molecule is bonded to an adjacent atom in a substantiallystraight series. Each atom can be bonded to an adjacent carbon atom by asingle-, double-, triple, or higher order-bond. By the term “branched”is meant a molecule comprising at least three atoms, any of which can bethe same or different, bonded in a substantially straight series,wherein the molecule further comprises at least one other atom, which isnot bonded to either of the terminal atoms of the substantially straightseries. Each atom can be bonded to an adjacent atom by a single-,double-, triple-, or higher order-bond. By “tetracycline” it is meant,the compound(4S,4aS,5aS,6S,12aS)-4-(dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4,4a,5,5a-tetrahydrotetracene-2-carboxamide.By “minocycline” it is meant, the compound(4S,4aS,5aR,12aS)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide.By “doxycycline” is meant the compound(4S,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide.By “oxytetracycline” it is meant, the compound(4S,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)-3,5,6,10,12,12a-hexahydroxy-6-methyl-1,11-dioxo-4,4a,5,5a-tetrahydrotetracene-).In a further aspect, there is provided a combination, compound,composition or use substantially as described herein with reference tothe accompanying figures and examples. There is provided a compoundcomprising a tetracycline, and at least one functional group comprisingN(O)n associated with the tetracycline; wherein n is an integer selectedfrom 1-3. Optionally, n is 1. Further optionally, the at least onefunctional group comprises NO. Still further optionally, the at leastone functional group comprises a nitroso group (—N═O). Alternatively,the at least one functional group comprises NO and is selected from adiazeniumdiolate molecule; a NONOate molecule (R₁R₂N—(NO—)—N═O; whereinR₁ and R₂ are each independently selected from alkyl groups); and athionitrite molecule (—SNO). Alternatively, n is 2. Optionally, the atleast one functional group comprises NO₂. Further optionally, the atleast one functional group comprises a nitro group (—NO₂).Alternatively, the at least one functional group comprises a nitrosooxy(—ONO) group. Further alternatively, the at least one functional groupcomprises NO₂ and is selected from arginine. Optionally, the at leastone functional group is L-arginine, and optionally acts as a substratefor nitric oxide synthase. Further alternatively, n is 3. Optionally,the at least one functional group comprises NO3. Further optionally, theat least one functional group comprises a nitrate group (—ONO₂), forexample, a nitrate ester, optionally a nitrate ester of an alcohol.Still further optionally, the at least one functional group comprises anitrate group (—ONO2), for example, a nitrate ester of an alkyl alcohol.Alternatively, the at least one functional group comprises a nitrategroup (—ONO2), for example, a conjugate base of nitric acid (nitrateion). By “associated with” is meant involving at least one chemicalinteraction. Optionally, the at least one functional group comprisingN(O)n is involved in at least one chemical interaction with thetetracycline. Further optionally, the at least one functional groupcomprising N(O)n involves at least one electrostatic interaction withthe tetracycline. Optionally, the at least one functional groupcomprising N(O)n forms at least one chemical bond with the tetracycline.Further optionally, the at least one functional group comprising N(O)nis a nitrate ester, which forms at least one chemical bond with thetetracycline. Optionally or additionally, the at least one functionalgroup comprising N(O)n forms at least one chemical bond with thetetracycline via a linker molecule. Further optionally, the at least onefunctional group comprising N(O)n forms at least one chemical bond withthe tetracycline via a linker molecule, wherein the at least onefunctional group comprising N(O)n is attached as a Mannich base to thetetracycline, optionally to the primary amide of the tetracycline.Optionally, the chemical bond is an ionic bond, wherein the interactionbetween the at least one functional group comprising N(O)n and thetetracycline is an interaction between oppositely charged atoms (orions). Optionally, the oppositely charged atoms (or ions) arerespectively located on or at the tetracycline and the at least onefunctional group comprising N(O)n. Preferably, the at least onefunctional group comprising N(O)n is selected from arginine and nitrateion (ONO3-). Alternatively, the at least one chemical bond is a covalentbond between the at least one functional group comprising N(O)n and thetetracyline. Optionally, the electrons are respectively located on or ateach of the at least one functional group comprising N(O)n and thetetracycline. Further optionally, the electrons are common (shared)electrons of the at least one functional group comprising N(O)n and thetetracycline, forming a covalent bond therebetween. Preferably, thecompound, or the at least one functional group, is capable of releasinga molecule comprising N(O)n; wherein n is an integer selected from 1-3.Optionally, the compound, or the at least one functional group, iscapable of releasing a molecule comprising NO (nitric oxide).Alternatively, the compound, or the at least one functional group, iscapable of releasing NO2 (nitrogen dioxide). Further alternatively, thecompound, or the at least one functional group, is capable of releasingNO3 (nitrate). By “capable of releasing a molecule” is meantdissociation of a molecule from the compound, such that the molecule isno longer associated with the tetracycline. Nitric oxide is a gaseousmolecule that is unsuitable for oral administration. There are severalpharmacologically relevant nitric-donor groups than are known to releasenitric oxide in response to conditions found in the human body afteradministration. Exemplary nitric-donor groups are described in “NitricOxide Donors: For Pharmaceutical and Biological Applications”; PengGeorge Wang, Tingwei Bill Cai, Naoyuki Taniguchi, Wiley (2005), which isincorporated herein by reference. Optionally, the tetracycline isdoxycycline. By “doxycycline” is meant the compound(4S,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide.Optionally, the at least one functional group comprising N(O)ncomprises: (a) a short-chain a short chain aza-alkyl, aza-alkenyl, oraza-alkynyl molecule, which can be linear, branched, or cyclic, andwhich can be substituted or unsubstituted; and (b) at least one groupcomprising N(O)_(n), wherein n is an integer selected from 1-3,hereinafter referred to as a nitric-oxide donor group. Optionally, theat least one nitric oxide donor group comprises (a) a short-chain ashort chain aza-alkyl molecule, which can be linear, branched or cyclic,and which can be substituted or unsubstituted; and (b) at least one atleast one nitric oxide donor group. Optionally, the at least one nitricoxide donor-group comprises an amine, which can be linear, branched orcyclic, and which can be substituted or unsubstituted. Furtheroptionally, the at least one nitric oxide donor group comprises asecondary amine, which can be linear, branched or cyclic, and which canbe substituted or unsubstituted. Optionally, the at least one nitricoxide donor group comprises an aza-ethyl molecule (ethyl amine) and atleast one at least one nitrate group. Further optionally, the at leastone nitric oxide donor group comprises an aza-diethyl molecule (diethylamine) and at least one at least one nitric oxide donor group.Preferably, the at least one nitric oxide donor group is a nitrate-estercontaining group. Optionally, the at least one nitric oxide donor groupcomprises an aza-diethyl molecule (diethyl amine), wherein one nitrategroup is attached to each ethyl group. Preferably, the at least onenitric oxide donor group is N,N-di-ethylnitrate amine. Optionally, theat least one nitric oxide donor group comprises a heterocyclic amine,which can be substituted or unsubstituted. Optionally, the at least onenitric oxide donor group comprises an aza-pentyl molecule and at leastone at least one nitrate group. Further optionally, the at least onenitric oxide donor group comprises an aza-cyclo-pentyl molecule and atleast one at least one nitrate group. Still further optionally, the atleast one nitric oxide donor group comprises a piperidine molecule andat least one at least one nitrate group. Optionally, the at least onenitric oxide donor group comprises a piperidine molecule and at leastone at least one alkyl-nitrate group. Further optionally, the at leastone nitric oxide donor group comprises a piperidine molecule and atleast one at least one methyl-nitrate group. Optionally, the at leastone nitric oxide donor group comprises a piperidine molecule and atleast one at least one alkyl-nitrate group. Further optionally, the atleast one nitric oxide donor group comprises a piperidine molecule andat least one at least one methyl-nitrate group. Optionally, the at leastone nitrate group, optionally the at least one alkyl-nitrate group, isattached to a carbon atom of the heterocyclic amine, optionally a carbonatom of the piperidine molecule. Further optionally, the at least onenitrate group, optionally the at least one alkyl-nitrate group, isattached to the carbon atom at position 3 of the heterocyclic amine,optionally the carbon atom at position 3 of the piperidine molecule.Optionally, the compound isamido-N—[N,N-diethylnitrate-aminomethyl]-α-6-deoxy-5-oxytetracycline.Alternatively, the compound isamido-N-[3-methylnitratepiperidinomethy]-α-6-deoxy-5-oxytetracycline.Further alternatively, the compound is 6-deoxy-5-oxytetracycline nitratesalt.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will be described, with referenceto the accompanying drawings, in which

FIG. 1 is a graph depicting MMP-9 activity in response to PMA, 150 □M ofdoxycycline, 450 □M of nitro amine and 150 □M of MJ3-53 (Manich basedinitrate);

FIG. 2 is a graph depicting MMP-2 activity in response to PMA, 150 □M ofdoxycycline, 450 □M of nitro amine and 150 □M of MJ3-53 (Manich basedinitrate); and

FIG. 3 is a graph depicting MMP-9 expression in patients with varyingdegrees of DHF.

FIG. 4 demonstrates the effect of SI1003, SI1004, SI1005 and doxycyclineon MMP-9 activity in PMA stimulated breast cancer cells (NC=negativecontrol).

FIG. 5 demonstrates the effect of SI1003, SI1004, SI1005 and doxycyclineon pro-MMP-2 activity in PMA stimulated breast cancer cells (NC=negativecontrol).

FIG. 6 demonstrates the effect of SI1003, SI1004, SI1005 and doxycyclineon MMP-2 activity in PMA stimulated breast cancer cells (NC=negativecontrol).

FIG. 7 demonstrates inhibition of MMP-2 and MMP-9 activity in responseto SI1005 (MJ-169).

FIG. 8 demonstrates inhibition of MMP-2 and MMP-9 activity in responseto SI1004 (MJ170).

FIG. 9 demonstrates the change in plasma MMP-9 levels from baseline to72 hours following the administration of doxycycline hyclate (Group 1),SI1004 (Group 2) and SI1005 (Group 3) to groups of 6 cynomolgus monkeys

FIG. 10. Effect of Doxycycline, SI1004 and SI1005 on colon cancer cellinvasiveness (NC=negative control; PC=positive control).

FIG. 11A. Impact of doxycycline hyclate (Doxy) and SI1004 on MMP-9 mRNAin TNFα treated human cardiac fibroblasts (n=3 per group). Shaded bar is0.1% DMSO+TNFα; striped bars are 0.1% DMSO+Doxycycline hyclate(concentrations shown) and solid black bars represent 0.1% DMSO+SI1004(concentrations shown). All values represent mean and SEM. I representsp<0.01 vs TNFα, **p<0.01 vs. Doxy. All bars were significantly elevatedvs. serum free controls.

FIG. 11B. Impact of doxycycline hyclate (Doxy) and SI1004 onproliferation of human cardiac fibroblasts (n=3 per group) following 72hours of serum starvation (clear bars) and subsequent exposure to 72hours of 2% fetal calf serum (FCS) with 0.1% DMSO (shaded bars), 0.1%DMSO+Doxycycline hyclate (concentrations shown, striped bars) and 0.1%DMSO+SI1004 (concentrations shown, black solid bars). All valuesrepresent mean and SEM. □ represents p<0.05 vs. 2% FCS, □□p<0.01 vs 2%FCS, *p<0.05 vs. Doxy, **p<0.01 vs. Doxy. All bars except SI1004 150 μMwere significantly elevated vs. serum free controls.

FIG. 12. Impact of doxycycline hyclate (striped bars) and SI1004 (solidbars) on [A] total MMP-9 and [B] total MMP-2 AUC in serum fromcynomologus monkeys following daily orogastric gavage dosing for 72hours (n=6). Doses used were 1.6 mg/kg doxycycline hyclate at time 0 and4.8 mg/kg doxycycline hyclate at 24 and 48 hours, or the molarequivalents of SI1004.

FIG. 13: Admixtures may be more effective than doxycycline inattenuating fibroblast proliferation, but not as effective as SI1004. Inthe following study Doxy and nitrate A are significantly better thanDoxy at inhibiting Cardiac Fibroblast Proliferation (p=0.011) at 150 uMHowever, Doxy and nitrate B are not (p=NS) at same concentration. SI1004is significantly more effective than doxycycline, Doxy and nitrate A,Doxy and nitrate B at 150 uM (all p<0.01).

FIG. 14. Admixtures reduce some inflammatory markers similarly to Doxy,e.g. IL-8. In the following study, Doxy and nitrate A can significantlyreduce IL-8 levels in TNFalpha stimulated PBMCs at 150 uM (p<0.01). Doxyalone and Doxy and nitrate B also reduce IL-8 levels compared tocontrols (p<0.05).

FIG. 15. Admixtures reduce some inflammatory markers more effectivelythan doxycycline, e.g. IL-1beta. In the following study, Doxy andnitrate A can significantly reduce IL-1 beta levels in TNFalphastimulated PBMCs (p<0.05). Doxy and nitrate B reduce IL-1 beta levels,but not significantly (p=NS).

FIG. 16. Admixtures reduce some inflammatory markers more effectivelythan doxycycline, e.g. IL-4. In the following study, Doxy and nitrate Acan significantly reduce IL-4 levels in TNFalpha stimulated PBMCs. Doxyand nitrate B admixtures reduce IL-4 levels, but not significantly(p=NS). IL-4 is reduced significantly more (p<0.01) by Doxy and nitrateA than either Doxy or Doxy and nitrate B. In this study, we see that notall NO donors provide similar efficacy.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will now be exemplified, withreference to the following non-limiting examples.

EXAMPLE 1 Preparation of Nitrate-Containing Group, N,N-DiethylnitrateAmine

All materials were purchased from the Sigma-Aldrich chemical company.With reference to Scheme 1, 1.5 mL fuming nitric acid was dissolved in10 mL DCM at −15° C. Diethanolamine (0.42 g, 4 mmole) dissolved in DCM(3 mL) was added dropwise over 20 minutes. The reaction mixture was thenleft stirring for a further 30 minutes before acetic anhydride (2 mL)was added to quench the reaction. The reaction was then left stirringfor a further 5 minutes to form a precipitate. The precipitate wasfiltered washed with cold DCM and dried under vacuum to giveN,N-diethylnitrate amine as a white solid.

HRMS ESI+ve C4H9N3O6 [M+H] requires 196.0570, found 196.0574. 1H NMR δ3.52-3.54 triplet (2×CH2-O), δ 4.81-4.83 multiplet (2×CH2-N).

EXAMPLE 2 Preparation of Nitrate-Containing Group, 3-MethylnitratePiperidine

With reference to Scheme 2, 1.5 mL fuming nitric acid was dissolved in10 mL DCM at −15° C. 3-hydroxymethyl piperidine (0.46 g, 4 mmole)dissolved in DCM (3 mL) was added dropwise over 20 minutes. The reactionmixture was then left stirring for a further 30 minutes before aceticanhydride (2 mL) was added to quench the reaction. The reaction was thenleft stirring for a further 5 minutes.

The pH of the reaction mixture was then adjusted to 14 with 7M NaOH. Thereaction mixture was then extracted with DCM (3×2o mL) and the combinedorganic extracts were washed with brine, dried over Na2SO4, filtered andsolvent removed in vacuo to give 4-methyl nitrate piperidine as a paleyellow oil.

HRMS ESI+ve C6H12N2O3 [M+H]+ requires: 161.0921; found: 161.0923. 1HNMR: δ 3.62 multiplet (CH2-ONO2), δ 3.35-3.20 multiplet (C2, C6 CH2),2.19 multiplet (C3CH) 2.11-1.90 multiplet (C5CH2).

EXAMPLE 3 Preparation ofAmido-N—[N,N-Diethylnitrate-Aminomethyl]-α-6-Deoxy-5-Oxytetracycline

Referring to Scheme 3, N,N-di-ethylnitrate amine (0.095 g, 0.487 mmole;as prepared in Example 1) and paraformaldehyde (0.016 g, 0.487 mmole)were suspended in 10 mL isopropyl alcohol and heated to 75° C. under aninert atmosphere for 30 minutes until a clear solution was obtained. Thereaction mixture was then cooled to 40° C. and doxycycline hyclate(0.250 g, 0.487 mmole), dissolved in a mixture of 5 mL isopropyl alcoholand 0.5 mL methanol, was added dropwise over 5 minutes. The reactionmixture was stirred at 40° C. for a further two hours. Upon completionof the reaction, the mixture was cooled and solvent removed to giveamido-N—[N,N-diethylnitrate-aminomethyl]-α-6-deoxy-5-oxytetracycline asa pale yellow solid.

MS ESI-ve C27H33N5O14 [M−H]− requires 650.1951, found 650.1938. 1H NMR:δ 4.05 ppm singlet (Mannich methylene), δ 7.5, 6.95 ppm triplets and7.85 ppm doublet (three phenyl protons) 2.9 ppm & 2.8 ppm singlets(dimethylamino, C4), δ 9.6 ppm singlet (amide), 3.52-3.54, triplet and4.81-4.83, multiplet (diethyl amino nitrate).

or alternative synthesis:

Amido-N-[Bis-(β-Nitrooxyethyl)Aminomethyl]-α-6-Deoxy-5-Oxytetracycline

Diethanolamine-dinitrate (234 mg, 1.2 mM, 1.2 eq), doxycycline free base(414 mg, 1 mM, 1.0 eq) and paraformaldehyde (60 mg, 2 mM, 2 eq) weredissolved in anhydrous tetrahydrofuran (10 ml) and heated to refluxingfor 2 h under nitrogen environment. Then another portionparaformaldehyde (60 mg, 2 mM, 2 eq) were added into the reactionmixture. After refluxing for another 2 h, the reaction mixture wascooled to room temperature and filtered. The filtrates were collectedand the solvent was removed. The resulting solids were dried undervacuum to afford the title compound as a brown microcrystalline solid(162 mg, 25%). m p.=101-104° C. Calculated for C₂₇H₃₂N₅O₁₄=650.2024;found (M−H)⁻=650.1965. ¹H NMR (400 MHz, d₆-DMSO) δ 15.2 (1H, s), 11.5(1H, s), 9.64 (1H, s), 9.1 (1H, s), 7.54 (1H, t, J=8), 6.94 (1H, d,J=8), 6.88 (1H, d, J=8), 5.4 (1H, s), 4.8 (4H, t, J=5), 4.65 (1H, dd,J=12, 7) 4.44 (1H, dd, J=12, 7) 4.17 (1H, s) 3.46-3.44 (5H, m) 2.73-2.65(7H, m) 2.50-2.52 (1H, m) 1.47 (3H, d, J=7). ¹³C NMR (400 MHz, d₆-DMSO)ppm: 192.5, 171.6, 161.1, 147.8, 136.6, 115.8, 115.6, 115.5, 107.2,73.2, 71.6, 68.8, 68.6, 68.0, 67.0, 62.0, 49.8, 45.2, 44.2, 41.3, 31.2;15.8.

IR (KBr) v (cm⁻¹): 3382; 2969; 1648; 1383; 1283; 849.

EXAMPLE 4 Preparation ofAmido-N-[3-Methylnitratepiperidinomethy]-α-6-Deoxy-5-Oxytetracycline

Referring to Scheme 4, to 6-deoxy-5-oxytetracycline hyclate (0.461 g,0.899 mmole) in anhydrous THF (10 mL) was added 3-methylnitratepiperidine (as prepared in Example 2) and 0.1 mL 37% formaldehydesolution. The reaction mixture was stirred at 40° C. for 16 hours. Thereaction mixture was then cooled and solvent removed under reducedpressure to giveamido-N-[4-methylnitratepiperidinomethy]-α-6-deoxy-5-oxytetracycline asa pale yellow solid. MS APCI C29H36N4O11 [M+NH4] requires 616.2831 found616.2944. ¹H NMR: δ 4.05 ppm singlet (Mannich methylene), δ 7.5, 6.95ppm triplets and 7.85 ppm doublet (three phenyl protons) 2.9 ppm & 2.8ppm singlets (dimethylamino, C4), δ 9.6 ppm singlet (amide), δ 3.62multiplet (CH₂—ONO₂), δ 3.35-3.20 multiplet (C2, C6CH2), 2.19 multiplet(C3CH) 2.11-1.90 multiplet (C5CH₂)

or alternative synthesis:

Amido-N-[3-(Nitrooxymethyl)Piperidinomethyl]-α-6-Deoxy-5-Oxytetracycline

3-Nitrooxymethyl piperidine (192 mg, 1.2 mM, 1.2 eq), doxycycline freebase (414 mg, 1 mM, 1.0 eq) and paraformaldehyde (60 mg, 2 mM, 2 eq)were dissolved in anhydrous tetrahydrofuran (10 ml) and heated torefluxing for 2 h under nitrogen environment. Then another portion ofparaformaldehyde (60 mg, 2 mM, 2 eq) was added into the reactionmixture. After refluxing for another 2 h, the reaction mixture wascooled to room temperature and filtered. The filtrates were collectedand the solvent was removed. The resulting solids were dried undervacuum to afford the title compound as a pale yellow microcrystallinesolid (153 mg, 25%). Calculated for C₂₉H₃₅N₄O₁₁=615.2308; found(M−H)⁻=615.2291. ¹H NMR (400 MHz, d₆-DMSO) δ 15.3 (1H, s) 11.6 (1H, s)10.12 (1H, s) 9.64 (1H, s) 7.54 (1H, t, J=8) 6.94 (1H, d, J=8) 6.88 (1H,d, J=8) 5.72 (1H, s) 4.65 (1H, dd, J=12.7) 4.45-4.30 (3H, m) 4.07 (1H,s) 3.22-3.35 (2H, m) 2.98-2.89 (2H, m) 2.80-2.65 (8H, m) 2.15-1.96 (2H,m) 1.89-1.75 (2H, m) 1.52-1.4 (4H, m).

EXAMPLE 5 Preparation of 6-Deoxy-5-Oxytetracycline Nitrate Salt

With reference to Scheme 5, a nitrate salt is prepared by adding silvernitrate (0.50 g, 2.96 mmole) to a solution of doxycycline hydrochloride(1.52 g, 2.96 mmole) in acetonitrile (20 mL). The solution is thenstirred at room temperature for 30 minutes.

After 30 minutes, a white precipitate of silver chloride was removed byfiltration to leave a pale yellow solution. This solution was addeddropwise to cold diethyl ether (100 mL) to form a pale yellowprecipitate that was filtered, washed with cold diethyl ether, and driedunder vacuum.

HRMS ESI C22H24N2O8 [M]+ requires 445.1605, found 445.1602.

EXAMPLE 6 Docycycline-5-Nitrate

A solution of doxycyline (414 mg, 1 mmol) in 5 ml THF was added to thesolution of Cu(NO₃)₂ (750 mg, 3 mmol) in 15 ml of acetic anhydride,which had been reacted for 2 h at room temperature. After reacted at−10° C. for 3 hour, the reaction mixture was filtered. The solvent ofthe filtrate was removed and dried under vacuum to give an amber solid(215, 44%). Calculated for C₂₂H₂₄N₃O₁₀=490.1456; found (M+H)⁺=490.1469.

EXAMPLE 7

4-Nitrooxymethyl piperidine (192 mg, 1.2 mM, 1.2 eq), doxycycline freebase (414 mg, 1 mM, 1.0 eq) and paraformaldehyde (60 mg, 2 mM, 2 eq)were dissolved in anhydrous tetrahydrofuran (10 ml) and heated torefluxing for 2 h under nitrogen environment. Then another portion ofparaformaldehyde (60 mg, 2 mM, 2 eq) was added into the reactionmixture. After refluxing for another 2 h, the reaction mixture wascooled to room temperature and filtered. The filtrates were collectedand the solvent was removed. The resulting solids were dried undervacuum to afford the title compound as a pale yellow microcrystallinesolid (128 mg, 21%). m p.=130-132° C. Calculated forC₂₉H₃₅N₄O₁₁=615.2308; found (M−H)⁻=615.2277. ¹H NMR (400 MHz, d₆-DMSO) δ15.3 (1H, s) 11.6 (1H, s) 9.64 (1H, s) 9.1 (1H, s) 7.54 (1H, t, J=8)6.94 (1H, d, J=8) 6.88 (1H, d, J=8) 5.72 (1H, s) 4.65 (1H, dd, J=12, 7)4.45-4.30 (3H, m) 4.07 (1H, s) 3.22-3.35 (2H, m) 2.98-2.89 (2H, m)2.73-2.65 (7H, m) 2.50 (1H, m) 2.15-1.96 (2H, m) 1.89-1.75 (2H, m)1.52-1.4 (5H, m). ¹³C NMR (400 MHz, d₆-DMSO) ppm: 192.5, 171.6, 161.1,147.8, 136.7, 115.8, 115.6, 115.5, 107.1, 76.4, 68.9, 68.2, 66.6, 50.7,45.3, 41.6, 38.4, 31.2, 26.6, 15.8.

IR (KBr) v (cm⁻¹): 3401; 29769; 1634; 1383; 1279; 867.

EXAMPLE 8

Amido-N-[4-nitrooxypiperidinomethyl]-α-6-deoxy-5-oxytetracycline

4-nitrooxypiperidine (175 mg, 1.2 mM, 1.2 eq), doxycycline free base(414 mg, 1 mM, 1.0 eq) and paraformaldehyde (60 mg, 2 mM, 2 eq) weredissolved in anhydrous tetrahydrofuran (10 ml) and heated to refluxingfor 2 h under nitrogen environment. Then another portion ofparaformaldehyde (60 mg, 2 mM, 2 eq) was added into the reactionmixture.

After refluxing for another 2 h, the reaction mixture was cooled to roomtemperature and filtered. The filtrates were collected and the solventwas removed. The resulting solids were dried under vacuum to afford thetitle compound as a pale yellow microcrystalline solid (192 mg, 32%).Calculated for C₂₈H₃₃N₄O₁₁=601.2151; found (M−H)⁻=601.2152. ¹H NMR (400MHz, d₆-DMSO) δ 15.3 (1H, s) 11.6 (1H, s) 9.64 (1H, s) 7.54 (1H, t, J=8)6.94 (1H, d, J=8) 6.88 (1H, d, J=8) 5.72 (1H, s) 5.27-5.30 (m, 1H) 4.65(1H, dd, J=12, 7) 4.43 (1H, dd, J=12, 7) 4.07 (1H, s) 3.22-3.25 (4H, m)2.73- 2.65 (7H, m) 2.50 (1H, m), 1.88-1.91 (2H, m) 1.47 (3H, d, J=7).

EXAMPLE 9

Amido-N-[3-(nitrooxymethyl)piperidinomethyl]-α-6-deoxy-5-oxytetracycline

3-Nitrooxymethyl piperidine (192 mg, 1.2 mM, 1.2 eq), doxycycline freebase (414 mg, 1 mM, 1.0 eq) and paraformaldehyde (60 mg, 2 mM, 2 eq)were dissolved in anhydrous tetrahydrofuran (10 ml) and heated torefluxing for 2 h under nitrogen environment. Then another portion ofparaformaldehyde (60 mg, 2 mM, 2 eq) was added into the reactionmixture. After refluxing for another 2 h, the reaction mixture wascooled to room temperature and filtered. The filtrates were collectedand the solvent was removed. The resulting solids were dried undervacuum to afford the title compound as a pale yellow microcrystallinesolid (171 mg, 28%). Calculated for C₂₉H₃₅N₄O₁₁=615.2308; found(M−H)⁻=615.2305.

EXAMPLE 10

Amido-N-[(β-nitrooxyethyl)aminomethyl]-α-6-deoxy-5-oxytetracycline

1-Methylaminoethyl nitrate (200 mg, 1.88 mM, 1.2 eq), doxycycline freebase (700 mg, 1.55 mM, 1.0 eq) and paraformaldehyde (93 mg, 3.1 mM, 2eq) were dissolved in anhydrous tetrahydrofuran (10 ml) and heated torefluxing for 2 h under nitrogen environment. Then another portionparaformaldehyde (93 mg, 3.1 mM, 2 eq) were added into the reactionmixture. After refluxing for another 2 h, the reaction mixture wascooled to room temperature and filtered. The filtrates were collectedand the solvent was removed. The resulting solids were dried undervacuum to afford the title compound as a pale yellow microcrystallinesolid (261 mg, 30%). Calculated for C₂₆H₃₁N₄O₁₁=575.1995; found(M−H)⁻=575.2032. ¹H NMR (400 MHz, d₆-DMSO) δ 15.2 (1H, s), 11.5 (1H, s),9.64 (1H, s), 9.64 (1H, s), 7.54 (1H, t, J=8), 6.94 (1H, d, J=8), 6.88(1H, d, J=8), 5.4 (1H, s), 4.8 (2H, t, J=5), 4.65 (1H, dd, J=12, 7) 4.44(1H, dd, J=12, 7) 4.17 (1H, s) 3.46-3.44 (3H, m) 2.73-2.65 (7H, m)2.50-2.52 (1H, m) 1.47 (3H, d, J=7).

EXAMPLE 11

Amido-N-[4-nitrooxypiperidinomethyl]-tetracycline

4-nitrooxypiperidine (175 mg, 1.2 mM, 1.2 eq), tetracycline free base(414 mg, 1 mM, 1.0 eq) and paraformaldehyde (60 mg, 2 mM, 2 eq) weredissolved in anhydrous tetrahydrofuran (10 ml) and heated to refluxingfor 2 h under nitrogen environment. Then another portion ofparaformaldehyde (60 mg, 2 mM, 2 eq) was added into the reactionmixture. After refluxing for another 2 h, the reaction mixture wascooled to room temperature and filtered. The filtrates were collectedand the solvent was removed. The resulting solids were dried undervacuum to afford the title compound as a brown microcrystalline solid(216 mg, 36%). Calculated for C₂₈H₃₃N₄O₁₁=601.2151; found(M−H)⁻=601.2147. ¹H NMR (400 MHz, d₆-DMSO) δ 15.3 (1H, s) 11.6 (1H, s)9.64 (1H, s) 9.64 (1H, s) 7.54 (1H, t, J=8) 7.1 (1H, d, J=8) 6.93 (1H,d, J=8) 5.27-5.30 (m, 1H) 5.10 (1H, s) 4.65 (1H, dd, J=12, 7) 4.43 (1H,dd, J=12, 7) 4.07 (1H, s) 3.22-3.25 (4H, m) 2.65-2.73 (7H, m) 2.50 (1H,m), 2.04-2.13 (2H, m) 1.88-1.91 (2H, m) 1.53 (3H, s).

EXAMPLE 12

Amido-N-[bis-(β-nitrooxyethyl)aminoethyl]-α-6-deoxy-5-oxytetracycline

Diethanolamine dinitrate (195 mg, 1 mmol, 1 eq) doxycycline free base(450 mg, 1 mmol, 1 eq) and acetaldehyde (110 uL, 88 mg, 2 eq) weredissolved in anhydrous tetrahydrofuran (10 ml) and heated to reflux for2 hours under nitrogen environment. A further 2 equivalents ofacetaldehyde were added to the reaction mixture and the reactioncontinued for a further 2 h. The reaction mixture was then cooled toroom temperature and filtered. THF was removed from the filtrate viarotary evaporation and the resultant residue was dried under vacuum togive an amber solid. (235 mg, 35%) ¹H NMR (400 MHz, d₆-DMSO) δ 15.2 (1H,s), 11.5 (1H, s), 9.64 (1H, s), 7.54 (1H, t, J=8), 6.94 (1H, d, J=8),6.88 (1H, d, J=8), 5.4 (1H, s), 4.57 (4H, t, J=5), 4.44, 1H, dd, J=12,7) 4.17 (1H, s) 3.46-3.44 (5H, m) 2.73-2.65 (7H, m) 2.50-2.52 (1H, m)1.78 (3H, d, J=7) 1.47 (3H, d, J=7).

EXAMPLE 13 & 14

Doxycycline-12a-nitrate; and minocycline-12a-nitrate; both of which maybe prepared by mild nitration under acidic conditions. In vitropharmacological evaluation

Cells (CaCo2 cells) were seeded onto a 12-well plate, and allowed togrow to 70% confluence. When cells were 70% confluent, the media on thecells were replaced with serum-free media. Cells were then treated withincreasing concentrations of test compound (50 □M-250 □M), for 3 hoursin a 37° C. incubator. After 3 hours, 10 □M PMA (Phorbol 12-myristate13-acetate) was added to the cells to induce production of MMPs. Cellswere incubated for 24 hours in a 37° C. incubator. After 24 hours, themedia from each well were collected and centrifuged at max speed for 5minutes to pellet any cellular debris, and the media was removed tofresh microfuge tubes. A Bradford assay was conducted to determine theprotein concentration of each media sample. An equal proteinconcentration of each media sample was loaded onto a zymography gel,which was run for 150V/2 hours. Following this, the zymography gel waswashed three times for 20 minutes in 2.5% Triton X Buffer and was washed2 times in zymography buffer before being incubated in zymography bufferat 37° C. for 48-72 hours to allow any MMP9 and MMP2 present to digestthe gelatinase in the gel. Following this, the gels were stained incoomassie blue stain for 3 hours with gentle rocking and destained for 1hour, resulting in a blue gel with clear bands where MMP's that werepresent had digested through the gelatine in the zymography gels.Densitometry analysis was performed to quantitate the amount of MMPspresent relative to the PMA positive control sample. Referring to FIG.1, addition of 150 uM of doxycycline did not affect MMP-9 levels.N,N-diethylnitrate amine, at equimolar concentrations to the Mannichbase dinitrate(amido-N—[N,N-diethylnitrate-aminomethyl]-α-6-deoxy-5-oxytetracycline)on its own, inhibited MMP-9 by over 50%. However, the combination ofdoxycycline with the nitrate amineamido-N—[N,N-diethylnitrate-aminomethyl]-α-6-deoxy-5-oxytetracycline](MJ3-53) suppressed MMP-9 activity by approximately 60%. Referring toFIG. 2, it can be seen that MMP-2 activity was significantly inhibitedby doxycycline (80%), and the combination withamido-N—[N,N-diethylnitrate-aminomethyl]-α-6-deoxy-5-oxytetracyclinereduced MMP-2 activity by about 40%. These data demonstrate that acompound of the present invention is capable of significantly alteringMMP expression, and finds utility in treating or preventing heartfailure, optionally heart failure caused by or associated with diastolicdysfunction; where MMP-9 levels are three times higher in the advancedstages compared with mild DHF. In DHF, MMP2 is 40 to 50% higher and MMP9is 200-300% higher in heart failure patients than in asymptomatichypertensive patients. In the present example, surprisingly, theamido-N—[N,N-diethylnitrate-aminomethyl]-α-6-deoxy-5-oxytetracyclineinhibits MMP9 more than by the constituent doxycycline andN,N-diethylnitrate amine. Moreover, the pattern of MMP 2 and MMP9inhibition may be more beneficial than doxycycline alone, which, inthese examples, did not reduce MMP9.

In-vitro/in-vivo Effects of Doxycycline and SI1004

The purpose of this study was to evaluate the in-vitro/in-vivo effectsof doxycycline and SI1004, a novel NO-releasing analogue of doxycyclinewhich could be applied to the treatment of disorders associated withelevated MMP-9 including ALVDD and HFpEF.

Methods

Direct Inhibition of Recombinant MMP-2 and MMP-9 with SI1004 andDoxycycline.

Nitrocycline, SI1004, a dinitroxyethyl conjugate with doxycycline wasprepared in-house using conventional chemical approaches andcharacterised by ¹H, ¹³C NMR, High Resolution Mass Spectroscopy and HighPerformance Liquid Chromatography. Doxycycline hyclate was obtained fromSigma-Aldrich Ireland. In order to determine the relative directinhibitory effects of SI1004 and doxycycline on MMP-2 and MMP-9 we usedhuman recombinant enzymes (R&D Systems, Ireland) with the syntheticbroad-spectrum fluorogenic substrate(7-methoxycoumarin-4-yl)-acetyl-pro-Leu-Gly-Leu-(3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl)-Ala-Arg-NH₂(R&D Systems, UK) as previously described (34).

Effects of SI1004 and Doxycycline on Human Ventricular CardiacFibroblast (HCF) proliferation and on TNF-α treated HCF MMP-2 and MMP-9transcription.

The impact of SI1004 and doxycycline on MMP-2 and MMP-9 transcriptionwas evaluated in primary HCFs purchased from ScienCell ResearchLaboratories. Cells were cultured in Dulbecco's modified eagles medium(DMEM) (Gibco), supplemented with 10% Fetal Calf Serum (FCS) (Gibco) andpenicillin-streptomycin antibiotics (Gibco) in a 5% CO₂ humidifiedincubator kept at 37° C. To investigate effects of test articles on cellproliferation, HCF cells were serum starved for 72 hours and thentreated with either 75 or 150 μM of test article in DMSO in 2% FCS for afurther 72 hours. Cell viability was measured using the CellTitre-GloLuminescent Cell Viability Assay (Promega) which measures ATP as anindicator of the number of metabolically active cells. To investigatethe relative effects of doxycycline and SI1004 on TNFα treated HCFtranscription of MMP-2 and MMP-9, cells were treated with 10 ng/mL humanrecombinant TNFα (R&D Systems) for 72 hours in the presence of 75 μM or150 μM of test article in DMSO. RNA was isolated using a NucleoSpin RNAII Kit (Macherey-Nagel). First strand cDNA synthesis was carried outusing SuperScript II RT (Invitrogen). QPCR primers were designed so thatone of each primer pair was exon/exon boundary spanning to ensure onlymature mRNA was amplified. The sequences of the gene-specific primersused are as follows; MMP-2, 5′-CACGTGACAAGCCCATGGGGCCCC-3′ (forward),5′-GCAGCCTAGCCAGTCGGATTTGATG-3′ (reverse);MMP-9,5′-GTGCTGGGCTGCTGCTTTGCTG-3′ (forward),5′-GTCGCCCTCAAAGGTTTGGAAT-3′ (reverse). QPCR reactions were normalizedby amplifying the same cDNA with GAPDH primers,5′-ACAGTCAGCCGCATCTTCTT-3′ (forward), 5′-ACGACCAAATCCGTTGACTC-3′(reverse). QPCR was performed using Platinum SYBR Green qPCRSuperMix-UDG(Invitrogen). Amplification and detection were carried out using theMx3000P System (Stratagene). The PCR cycling program consisted of 40three-step cycles of 15 seconds/95° C., 30 seconds/TA and 30 seconds/72°C. Each sample was amplified in duplicate. In order to confirm signalspecificity, a melting program was carried out after the PCR cycles werecompleted. The samples were quantified by comparison with a standardcalibration curve created at the same time and the data was normalizedby an internal control (glyceraldehyde 3-phosphate dehydrogenase).

Effects of SI1004 and Doxycycline on MMPs, TIMP-1 and InflammatoryMarkers in Human Peripheral Blood mononuclear cells (PBMC) stimulatedwith TNF-α.

To further explore the relative impact of SI1004 and doxycycline oninflammatory cells (PBMC), venous blood (30 mL) was collected from threehealthy volunteers (age 30-37) in 10 mL S-Monovette tubes withanti-coagulant 9NC (Sarstedt). The blood was mixed with an equal volumeof D-PBS (Gibco) and two volumes of the mixture were layered over onevolume of Lymphoprep gradient solution (Axis-Shield). PBMC were isolatedby centrifugation at 400 g for 40 minutes. PBMC were collected from theplasma/lymphoprep interface and washed three times in D-PBS/0.1% BSA/2mM EDTA. PBMC were suspended at 1×10⁶ cells/mL in pre-warmed RPMI1640/10% FCS/2 mM L-glutamine/100 μg/mL penicillin G/100 μg/mLStreptomycin (all from Gibco). Cells (0.2×10⁶) were plated at aconcentration of 1.0×10⁶ in 96-well plates in duplicates, stimulatedwith 10 ng/mL TNFα (R&D Systems) with/without doxycycline hyclate orSI1004 (at 75 and 150 μM) and incubated for 24 hours at 37° C. On thefollowing day, all samples were centrifuged and supernatants were storedat −80° C. for immunoassays. Percent PBMC viability following drugtreatment was determined using the CellTiter-Glo Luminescent CellViability Assay (Promega) according to the manufacturer instructions.The cytokine profile of the cell supernatants was analysed using anultra-sensitive immunoassay with electrochemiluminescence detectionaccording to the manufacturer's instructions (Meso Scale Discovery). MMPsecretion was also quantified using multiplex immunoassays withelectrochemiluminescence detection as instructed by the manufacturer(MMP2/10 Duplex and MMP1/3/9 Triplex assays—MesoScale Discovery).Single-plex assays were used for monocyte chemotactic protein (MCP)-1(Meso Scale Discovery). Plates were analyzed using a Meso ScaleDiscovery Sector Imager 2400 instrument. Secreted TIMP-1 was quantifiedusing a standard ELISA (Amersham, GE Healthcare). TH1/TH2 10-plex assaywas used to study IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70,IL-13, and TNFα. The sensitivity (lowest level of detection) of theassays was 0.12 ng/mL and 0.1 ng/mL for MMP-2 and MMP-9, respectively.The coefficient of variation of the lower limit of the standard curvefor MMP-2 and MMP-9 was 4.9% and 1.2% respectively. Plates were analyzedusing a Meso Scale Discovery Sector Imager 2400 instrument.

Relative Effects of SI1004 and Doxycycline on Total MMP-2 and MMP-9Levels on Acute and Repeated Oral administration over three days withdose titration following day one in non-human primates (NHP).

A total of 12 purpose bred, purpose bred, naïve, non-human primates(cynomolgus monkeys, 2.9-4 kg) were sourced and randomly allocated in aparallel group design (n=6 per group) to receive SI1004, SI1005 andequimolar doses of doxycycline daily (1.6 mg/kg doxycycline hyclateequivalents, on day 1 and 4.8 mg/kg doxycycline equivalents on days 2and 3) by oral gavage in aqueous vehicle over a 3 day period. Studieswere carried out consecutively in two contract research organizationsites (Charles River, Sparks, Nev., US and Charles River, Shanghai,China). The study protocol was approved by PCS-SHG Institutional AnimalCare and Use Committee before conduct. During the study, care and use ofanimals was conducted in accordance with the guidelines of the USANational Research Council and the Canadian Council on Animal Care. Thecynomolgus monkey was chosen for this study in order to maximize thelikelihood of identifying responses that are similar to those that maybe expected in humans. Each animal was identified by a cage label andbody tattoo and was acclimated to orogastric dosing on at least twooccasions prior to the initiation of dosing. The vehicle (1% (w/v) tween80 and 0.5% (w/v) carboxymethylcellulose in deionized water) or 1.6mg/kg doxycycline hyclate (0 hours) or 4.8 mg/kg doxycycline hyclate(24, 48 hours) or the molar equivalent(s) of SI1004 or SI1005 wereadministered using an orogastric tube inserted through the mouth andadvanced into the stomach. The animals were temporarily restrained (i.e.manually) for dose administration, and were not sedated. Disposablesterile syringes and orogastric tubes were used for each animal/dose.Each dose was followed by a tap water flush of approximately 5 mL. Bloodsamples and blood pressure measurements were taken at the followingtimepoints: pre-dose (0 hours) and at 2, 4, 6, 12, 24, 26, 30, 36, 48,50, 54, 60 and 72 hours after first administration of test article. Wehave previously demonstrated an acute phase response in this model torepeated venepuncture (3-6 fold increase in high sensitivity C-reactiveprotein from baseline at 12 and 24 hours post dose respectively, (bothp=0.01 vs baseline), data not shown). Blood (300 μL) for serumpreparation was collected intoBD Vacutainer®+Serum SST™ tubes toaccelerate clotting 20 minutes prior to centrifugation to allow completeclotting to occur and centrifuged at 1500-2200 rpm at 2-8° C. for 10-15minutes. Under these conditions blood cells containing MMP, principallyneutrophils and platelets, undergo full degranulation. Since artifactualelevation of MMP-9 was an unavoidable feature of repeated venipuncturein our model, it was logical to stimulate full MMP-9 release duringsample collection. This provided greater inter-animal reproducibilityand a more dynamic analytical range for assessing the relative effectsof the test articles. Subsequent MMP-9 values provide an index of totalMMP-9 including circulating enzyme, amplified by repeated venipuncture,along with the cellular load released from storage granules duringclotting. The latter is influenced by earlier inflammatory signaling,transcription and storage. The serum was transferred to a cryovial andimmediately stored at −70° C. until analyzed for MMP-2 and MMP-9 via aLuminex ELISA (total MMP-2 and MMP-9) within 48 hours of collection. Theanalysis of each time point was repeated within 5 days. Values thatdiffered by more than 15% were repeated. The primary study endpoint wasthe change in plasma MMP-2 and MMP-9 levels at 72 hours. Secondaryendpoints were area under the curve (AUC) values of MMP-2 and MMP-9 overthe following periods: 0-24, 0-48 and 0-72 hours. Additional 0.4 mLaliquots were placed in K₂EDTA tubes and processed to plasma forcombined nitrate/nitrate (NO_(x)) analysis using a modified Greiss assayas previously described (35). Simultaneous blood pressure measurementswere made in triplicate using a femur cuff linked to an automated Omronanalyzer. Data are presented as mean±standard error of the mean (SEM)for continuous normal variables, median, interquartile range (IQR) with95% confidence intervals for non-normal continuous variables andfrequencies and percents for nominal/categorical variables. Comparisonsbetween doxycycline and SI1004 groups in the NHP study were made onchanges over the study period using independent two-sample t-tests forcontinuous normally distributed data, Mann-Whitney for skewed continuousand chi-squared for categorical data. Within group tests, comparingbaseline to 24, 48 and 72 hour values, were conducted using pairedsample t-tests and paired sample Wilcoxon tests where appropriate.Analyses were carried out using SPSS V.13 statistical software(Statistical Package for the Social Sciences: SPSS Inc, Chicago, Ill.,2001).

Results

Effects of Doxycycline and SI1004 on Activity of Recombinant Human MMP-2and MMP-9

Doxycycline and SI1004 had similar direct inhibitory effects on MMP-2and MMP-9 enzymatic activity. Doxycycline (100 μM) inhibited recombinanthuman MMP-2 (34.0±3.5%) and MMP-9 (33.3±3.5%) (p<0.05). Similarly SI1004(100 μM) inhibited MMP-2 and MMP-9 by 29.7±2.1% and 26.6±1.7%respectively (p<0.05). However, there was no direct inhibition of eitherenzyme by the test articles at 10 μM. These values suggest weak,non-selective inhibition of both gelatinases at enzyme level and areconsistent with doxycycline's low binding affinity for the MMPs.

Effects of Doxycycline and SI1004 on Human Cardiac Fibroblasts

In contrast to doxycycline hyclate, SI1004 significantly inhibited TNFαinduced upregulation of MMP-9 mRNA (p=0.01, FIG. 1A). MMP-9 proteinlevels were below the lower limit of quantification in doxycyclinehyclate or SI1004 treated cell supernatants. There were no significanteffects of doxycycline hyclate or SI1004 on MMP-2 mRNA expression. Also,unlike doxycycline, SI1004 (75-150 μM) caused significant inhibition ofHCF proliferation in 2% FCS following serum starvation for 72 hours,(p=0.02, FIG. 1B).

Effects of Doxycycline and SI1004 on Markers of Inflammation andCollagen Turnover in Human Peripheral Blood Mononuclear Cells

The effects of doxycycline hyclate and SI1004 on MMPs, TIMP-1,inflammatory cytokines and MCP-1 are presented in Table 1. Bothcompounds significantly inhibited PBMC supernatant MMP-9, TIMP-1, IFNγ,IL-8, IL-12p70 and MCP-1 (all p<0.05). SI1004 (150 μM) but notdoxycycline, inhibited IL-1β production at 150 μM (p=0.03). Doxycyclineinhibited TIMP-1 to a greater extent than SI1004 at both concentrations(p<0.05) and doxycycline, but not SI1004, inhibited MMP-3 (p<0.02).

Plasma MMP-2 and MMP-9 Levels Over 72 Hours with Daily Dosing ofDoxycycline and SI1004 in Cynomolgus Monkeys

Oral administration of SI1004 caused more effective suppression of totalserum MMP-9 protein levels than doxycycline (FIG. 12A). Between-groupdifferences were significant by day 2 and remained significant on day 3in terms of AUC (24-48 and 48-72 hours) and also in terms of MMP-9change from baseline at 48 and 72 hours (all p<0.05). Total MMP-2 levelswere similar over the 3-day treatment period (FIG. 12B). Maximum plasmadoxycycline concentration (Cmax) was noted on day 3 of dosing whereplasma doxycycline concentration achieved 5.1 μM (base equivalents).SI1004 caused an increase in mean plasma nitrite/nitrate (NOx) over theduration of the dosing period, with peaks at 6 hours post-dosing (i.e.at 6, 30 and 54 hours) consistent with activation of the SI1004 nitrategroup and NO release. NOx Cmax (μg/mL) for SI1004 was 12.1±2.2,47.9±2.2, and 50.4±12.5, on days 1, 2 and 3 respectively (all at 6 hourspost dose). Although the mean systolic blood pressure was higher in thedoxycycline hyclate group (109.7±7.1 mmHg vs 101±6.3, p<0.01), there wasno difference in diastolic blood pressure (58.6±6.0 mmHg vs 56.4±3.8mmHg, p=NS) and the pattern of NO release was not associated withsignificant differences in blood pressure (either systolic or diastolic)or heart rate at any time point.

TABLE 1 Impact of doxycycline hyclate and SI1004 on MMPs, TIMP-1,interleukins and MCP-1 protein levels in supernatants of PBMC treatedover 24 hours (n = 3). All values are mean ± SEM (ng/mL) DoxycyclineDoxycycline SI1004 SI1004 Control (150 μM) (75 μM) (150 μM) (75 μM)No.(%)/Mean ± SD No.(%)/Mean ± SD Interleukin-1β 40.8 ± 6.2  30.6 ± 5.0 34.6 ± 5.8  24.6 ± 1.2

  27.8 ± 6.6 Interleukin-4 12.0 ± 0.6   9.8 ± 0.8  9.4 ± 1.2  9.6 ± 0.6

  10.8 ± 0.2 Interleukin-5  49.8 ± 11.6  31.2 ± 5.8  40.8 ± 9.0  35.6 ±6.4   35.8 ± 8.4 Interleukin-8 15322 ± 264  6352 ± 1438

 8852 ± 2568  8826 ± 1364

10960 ± 484 Interleukin-10 121.2 ± 51.2  92.2 ± 33.8 136.6 ± 74.2 142.0± 55.2  125.6 ± 32.2 Interleukin-12p70 19.8 ± 1.0  14.2 ± 2.0

 16.2 ± 2.4

 15.2 ± 1.0

  16.6 ± 1.2

Interleukin-13 106.6 ± 0.6   80.2 ± 3.2  79.6 ± 17.2  89.4 ± 13.2   80.8± 24.8 MCP-1  598 ± 338  30.0 ± 8.0

 92.0 ± 54.0

 40.0 ± 14.0

 146 ± 54

Interferon γ 124.4 ± 6.8   86.6 ± 8.8

 99.5 ± 15.0

100.2 ± 7.6

 102.8 ± 4.8

MMP-1  262 ± 132  134 ± 48 144.0 ± 80.0   171 ± 66.0  208 ± 102 MMP-2 126.0 ± 104.6  96.8 ± 80.6  61.4 ± 54.2  95.4 ± 52  120.4 ± 86.4 MMP-311.3 ± 2.8   2.9 ± 0.4

 1.5 ± 1.5

 9.1 ± 6.1*   8.7 ± 5.2* MMP-9 29.4 ± 7.6   1.3 ± 0.6

 3.4 ± 1.6

 8.4 ± 2.1

**   18.4 ± 7.3

* MMP-10  84.4 ± 25.2  47.6 ± 15.8  36.0 ± 14.0  29.0 ± 14.6   26.6 ±18.4 TIMP-1 56.0 ± 4.2   6.8 ± 2.4

 12.6 ± 1.6

 22.0 ± 9.2

*   32.4 ± 9.4

* All values represent mean and SEM.

 represents p < 0.05 vs. TNFα treated controls,

 p < 0.01 vs TNFα treated controls, *p < 0.05 vs. Doxy, **p < 0.01 vs.Doxy. Abbreviations: MCP = monocyte chemotactic protein, MMP = matrixmetalloproteinase, TIMP = tissue inhibitor of matrix metalloproteinase.

SI1004 and doxycycline have low binding capacity to MMP-2 and MMP-9enzymes at concentrations achieved in-vivo. Both compounds inhibit TNFαinduced MMP-9, TIMP-1, IFNγ, IL-8, IL-12p70 and MCP-1 expression inPBMC. Unlike doxycycline, SI1004 inhibits IL-1β and also TNFα inducedMMP-9 mRNA in HCF and HCF proliferation. SI1004 has similar effects onMMP-2 in-vivo and more effectively reduces total plasma MMP-9 (medianAUC 4.3 μg/mL.hour, IQR 3.1-5.5) than doxycycline (median AUC 8.7μg/mL.hour, IQR 7.3-11.3, p<0.05 vs doxycycline) in NHPs.

Conclusions: This study demonstrates that doxycycline and SI1004 areimmunemodulatory MMP inhibitors. SI1004 provides more effectiveinhibition of inducible MMP-9 than doxycycline.

Discussion

HFpEF accounts for 40-60% of all cases of HF and is set to increase withcontinued high prevalence of ALVDD driven principally by hypertensionand diabetes. Experience to date with renin-angiotensin-aldosteronesystem (RMS) modifying therapies suggests that novel therapeuticapproaches are needed. While RMS modifying therapies have shownanti-fibrotic effects, several lines of in-vitro and in-vivo evidencepoint to co-existing inflammation and ECM remodeling as key drivers ofHFpEF pathophysiology. ECM remodeling is regulated by myocardial MMPsand TIMPs which have been elusive pharmacological targets in the clinic.The present study provides a pharmacological and pathophysiologicalrationale for further evaluation of immunomodulatory, broad-spectrum MMPinhibitor doxycycline and its novel NO-releasing analogue (SI1004) ascomponents of an anti-remodeling strategy in ALVDD and HFpEF.Furthermore, SI1004 reduces transcription of inducible myocardial MMP-9and total MMP-9 in-vivo more effectively than doxycycline and this mayprovide efficacy and safety advantages in chronic therapy. Abnormalitiesin the cardiac interstitium are central to the pathophysiology of ALVDDand HFpEF. These abnormalities include delayed relaxation, impaired leftventricular filling and/or increased stiffness in the myocardium.Myocardial remodeling is characterized by inflammation, fibrosis(increased collagen production, reduced collagen breakdown, alterationsin the relative balance of collagen I/III, changes in the biomechanicalproperties of myocardial collagen) and alterations in other componentsof the ECM such as fibronectin, laminin and elastin. Modulation of thecardiac interstitium in pressure/volume overload is partially regulatedby MMPs and TIMPs and recent human studies have associated serum andtissue myocardial MMP levels with increased arterial stiffness inpatients with hypertension, hypertrophic obstructive cardiomyopathy,diastolic dysfunction and HFpEF. Supporting these observations areanimal studies showing MMP-9 and its tissue inhibitor, TIMP-1, areassociated with the transition from hypertrophy to HF the development ofdiastolic dysfunction and HFpEF in models of chronic pressure-overload.MMP-2 and MMP-9 knockout mice develop less marked cardiomyocytehypertrophy and fibrosis following transverse aortic banding andpharmacological MMP inhibition prevents ventricular remodeling and HF inpressure overload states, including HF induced by inflammatorycytokines. However, direct pharmacological inhibition of MMPs has beenunsuccessful as a chronic therapy in the clinic. Over 60 MMP-bindinginhibitors have been tested, primarily in cancer and heart disease, withconsistently disappointing efficacy or unacceptable side-effectprofiles. The 24 human MMPs and their TIMPs also contribute to a largearray of important physiological processes. Thus, for example, chronic,direct inhibition of collagenases may actually facilitate myocardialfibrosis in pressure overload states. It is important to note that MMP-2has collagenase activity and activates other collagenases, unlike MMP-9,suggesting it may have role in the attenuation of excess collagendeposition in the myocardium. Conversely, MMP-9 basal activity isnormally low but its gene contains binding sites for AP-1, NF-κB, Sp-1,Ets-1 and Egr-1. Global deletion of MMP-9, endows mice with a benignphenotype in the absence of pathophysiological stress. However,following induction of myocardial infarction, MMP-9 knockout micedemonstrate reduced macrophage infiltration, left ventricular dilationand collagen accumulation as well as increased vascularity andperfusion. Taken together, these data indicate that pharmacologicalattenuation of inducible myocardial MMP-9 and MMP secretion withoutchronic direct enzyme inhibition could be an effective and/or safertherapeutic approach in patients with ALVDD and HFpEF. Doxycycline isthe only therapy licensed for human use as a MMP inhibitor, in thesetting of periodontal disease, and is currently under investigation byour group in ALVDD and HFpEF (EudraCT number: 2010-021664-16). As wellas direct inhibitory effects on a range of MMPs, doxycycline alsoinhibits the acute phase MMP-9 release from tertiary granules inneutrophils. The present study suggests that doxycycline has low bindingcapacity for myocardial MMP at plasma levels achieved in this study andduring chronic human dosing (<10 μM) and this may be an advantage interms of long term safety at conventional doses. Furthermore, the effectof doxycycline and SI004 on IFNγ and IL-12p70 secretion by TNFαstimulated PBMCs suggests a reduced capacity to promote T cellactivation. Both agents also suppress IL-8 and MCP-1 secretion fromactivated PBMCs indicating an ability to inhibit neutrophil and monocytechemotaxis. These data are in accordance with previous in-vivo evidenceof doxycycline suppression of neutrophil and cytotoxic T cellaccumulation in the aortic wall of patients undergoing electiveaneurysmal repair. Given the emerging importance of inflammation in theearly phases of HFpEF and the potentially causal role of MCP-1 in therecruitment of monocytes and initiation of interstitial fibrosis inanimal models of pressure overload our data suggest a beneficialanti-inflammatory role for doxycycline and SI004 in ALVDD and HFpEF. Theadditional effects of NO release on pro-inflammatory stimuli andgelatinase activity may amplify doxycycline's inhibitory effect in thesetting of HFpEF. Doxycycline reduces NO and peroxynitrite levels inmultiple cell types stimulated with inflammatory cytokines, partlythrough inducible nitric oxide synthase (iNOS) inhibition. It is alsoknown that intracellular NO formation can suppress IL-1β by inhibitingcaspase-1, the IL-1β converting enzyme which may explain the significantreduction of SI1004 on this inflammatory cytokine. NO can also affectthe cellular distribution and compartmentalization of MMP-9, decreaseMMP-9 mRNA stability and inhibit its transcription via effects on AP-1,NF□B and PEA3 promoter activity. Furthermore, vascular NO is depleted inhypertension and NO has well-known effects on vascular smooth musclecells, activating guanylate cyclase and increasing the formation ofcyclic guanosine monophosphate (cGMP), causing vasorelaxation, reducedpulse wave reflection and reduced central aortic pressure. NO and cGMPreleasing substances are associated with an improvement in diastolicrelaxation that suggest a beneficial effect in diastolic HF. A finalpotential advantage of nitrocycline is that while short and long-termuse of doxycycline can cause gastro-eosophageal irritation, NO isgastroprotective and NO donor groups can increase the intestinaltolerability and safety of a number of drugs. The present studyidentifies a number of key differences between SI1004 and its parentmolecule. Of potential importance in myocardial remodeling is thatSI1004 has superior efficacy on MMP-9 mRNA in TNF□ stimulated HCF.SI1004 may have less inhibitory effects on TIMP-1 and MMP-3 which areassociated with the attenuation of myocardial remodeling and increasedscar volume after myocardial injury. By processing samples to serum withcomplete clotting, which causes degranulation of PBMCs and platelets, weobtained an index of total MMP-9 protein in-vivo. SI1004 was strikinglymore effective than doxycycline hyclate in the inhibition of MMP-9,consistent with inhibitory effects on MMP-9 RNA and a broaderanti-inflammatory profile. These effects may make the nitrocyclineapproach therapeutically relevant in pathologies where there is a stronginflammatory component associated with elevated MMP-9 levels includingALVDD and HFpEF. In conclusion, ALVDD and HFpEF are diseases driven byinflammation, fibrosis and abnormalities of ECM turnover. This studypresents in-vitro and in-vivo evidence of efficacy of doxycycline andSI1004, a novel, NO-releasing tetracycline analogue, asimmunomodulatory, MMP inhibitors. SI1004 is a more effective inhibitorof MMP-9 transcription and serum MMP-9 in NHPs, than doxycycline. Theseagents are considered to be useful in treatment of diseases associatedwith elevated MMP.

Cancer Applications

As discussed in the background section, Matrix Metalloproteinase (MMP)levels in the plasma are known biomarkers of breast, colorectal, renal,pancreas, bladder and lung cancers (see Table 2).

TABLE 2 Candidate MMP and ADAM Biomarkers of Cancer (Roy, Yang et al.2009) Type of Cancer and MMPs/ADAMs Detected in Tissue/Body Fluid BreastMMP-13 Tissue MMP-9, TIMP-1 Serum, tissue MMP-9 Urine, serum, plasma,tissue ADAM12 Urine ADAM17 Tissue MMP-1 Tissue, nipple aspiratesPancreas MMP-9 Pancreatic juice, serum MMP-2 Pancreatic juice, tissueMMP-7 Tissue, plasma ADAM9 Tissue Lung VMMP-9, TIMP-1 Serum, bronchiallavage MMP-7 Tissue MMP-1 Tissue Bladder MMP-9 Tissue MMP-9, MMP-2 UrineMMP-9 Urine MMP-9, telomerase Urine Colorectal MMP-2 Tissue, plasmaMMP-9 Tissue MMP-2, MMP-9 Plasma MMP-7 Serum MMP-1 Tissue MMP-13 TissueOvarian MMP-9 Tissue MMP-9, MMP-14 Tissue MMP-2 Tissue MMP-2, MMP-9,MMP-14 Tissue ADAM17 Tissue Prostate MMP-2, MMP-9 Plasma, tissue MMP-2Tissue MMP-9 Urine ADAM8 Tissue ADAM9 Tissue Brain MMP-2 Tissue MMP-9Tissue MMP-2, MMP-9 Tissue, cerebrospinal fluid, urine

MMPs are involved in cancer cell intravasation and extravasation. Theyeffect Extracellular Matrix (ECM) degradation and disrupt cell-cellinteractions promoting cell migration. MMP-9 is also involved inendothelial-mesenchymal-transition (EMT) whereby cells acquire migratorycharacteristics and this is also facilitated by MMP-3 (via interactionswith E-cadherin and Rac1b). MMPs modulate growth factors and receptors.MMP-9 modulates vascular endothelial growth factors which promotestumour growth and angiogenesis. MMP-3 modulates insulin like growthfactor binding proteins and basic fibroblast growth factors and is alsoknown to activate MMP-9. MMPs also modulate tumour associatedinflammation (e.g. MMP-9 is involved in breast cancer inflammation) viacytokines and their receptors.

Anti Cancer Effect of the Compounds of the Invention

SI1004 (MJ-170, Dinitrate MB) is a more effective MMP-9 inhibitornitrocycline than SI1005 (MJ-169, Piperidine Mono MB), which has beenshown to inhibit MMP-3. Accordingly, it may be able to more selectivelyreduce MMP-9 protein levels than SI1004. Both SI1004 and SI1005 are morepotent MMP-9 inhibitors than conventional doxycycline.

In Vitro Data

Using in vitro breast cancer cell models (HT1080 cells), stimulated witha pro-inflammatory insult (PMA) to stimulate the over-production ofMMP-9, we see that doxycycline (Doxy), SI1004 (MJ-170, Dinitrate MB) andSI1005 (MJ-169, Piperidine Mono MB) all reduce MMP-9 production at 100micromolar concentrations. Using the same in vitro breast cancer cellmodels for examining MMP-2, we see that PMA reduces pro-MMP-2 anddoxycycline (Doxy), SI1004 (MJ-170, Dinitrate MB) and to a lesser extentSI1005 (MJ-169, Piperidine Mono MB) all reduce pro-MMP-2 production at100 micromolar. However, surprisingly, doxycycline (Doxy) also appearsto increase the conversion of available pro-MMP-2 to active MMP-2 (FIG.5). This, potentially, could be a concern for chronic doxycyclinetherapy in the treatment of cancer. Advantageously, we do not see thesame activation of MMP-2 with nitrocyclines.

Using models of direct enzyme inhibition, it is shown below that SI1004(MJ-170, Dinitrate MB) and SI1005 (MJ-169, Piperidine Mono MB) are morepotent inhibitors of MMP-9 than doxycycline. The 1050 value (μM) ofSI1005 (MJ-169, Piperidine Mono MB) for MMP-2 and MMP-9 are 63 (46-84)and 139 (86-223) respectively. The IC₅₀ value (μM) of SI1004, (MJ-170,Dinitrate MB) for MMP-2 and MMP-9 are 9.4 (8.5-10.4) and 25 (19-32)respectively. These are more potent than doxycycline which has anapproximate IC₅₀ value (μM) for MMP-2 and MMP-9 of 129 and 164respectively

TABLE 2 IC₅₀ values for the inhibition of MMP-2 and MMP-9 in response toSI1004, SI1005 and doxycycline. SI1004 (MJ-170) SI1005 (MJ-169)Doxycycline MMP-2 9.4 μM  63 μM 129 μM MMP-9  25 μM 139 μM 164 μM

MMP-8: SI1005 (MJ-169, Piperidine Mono MB) has around 53.8% inhibitionat 100 μM and 16.9% inhibition at 10 μM. SI1004 (MJ-170, MJ-170,Dinitrate MB) has around 60.7% inhibition at 100 μM and 26.0% inhibitionat 10 μM. Doxycyline has around 42.7% inhibition at 100 μM. MMP-13:SI1005 (MJ-169, Piperidine Mono MB) has around 28.4% inhibition at 100μM and 6.6% inhibition at 10 μM and SI1004 (MJ-170, MJ-170, DinitrateMB) has around 74.5% inhibition at 1000 and 46.0% inhibition at 10 μM.Doxycyline has around 54% inhibition at 100 μM. MMP-1: SI1005 (MJ-169,Piperidine Mono MB) has around 22% inhibition at 100 μM and 13%inhibition at 10 μM while SI1004 (MJ-170, MJ-170, Dinitrate MB) hasaround 63% inhibition at 100 μM and 19% inhibition at 10 μM. Doxycylinehas around 12% inhibition at 100 μM.

In Vivo Data

Nitrocycline compounds SI1004 (MJ-170, Dinitrate MB, Group 2), SI1005(MJ-169, Piperidine Mono MB, Group 3) and doxycycline hyclate control(Doxy Group 1) were administered to cynomolgus monkeys (n=6 per group)as described in the method below. The test articles were administered byoral gavage once daily for three days (Doxycycline hyclate 1.6 mg/dayand equimolar doses of the nitrocyclines were administered on Day 1.These doses were equivalent to 100 mg/day of doxycycline base. The doseof doxycycline hyclate was increased to 4.6 mg/kg on the second andthird 20 day. Equimolar doses of nitrocyclines were administered. Thisdose was equivalent to a 300 mg/day dose of doxycycline base). Theprimary endpoint of this study was the changes in MMP-9 from baseline to72 hours. In the high dose doxycycline group (Group 1), MMP-9 levelsincrease. In the SI1004 (Group 2) MMP-9 levels are significantly reducedcompared to doxycycline. In the SI1005 (Group 3) MMP-9 levels aresignificantly reduced compared to doxycycline and SI1004. These dataprovide proof-of-concept in vivo support for the use of SI1004 andSI1005 as more potent inhibitors of MMP-9 compared to doxycycline.Furthermore, SI1004 and SI1005 are more potent inhibitors ofinflammatory cytokines such as IL-1b, IL-4 and IL-8 compared todoxycycline (data not shown). Finally, in order to provide a functionalmodel of tumour cell invasion, the following data show that that at lowdose, doxycycline (Doxy) does not reduce colon cancer cell invasiveness,whereas SI1004 (MJ-170, Dinitrate MB) and SI1005 (MJ-169, PiperidineMono MB) do (FIG. 10). Overall nitrocyclines SI1004 and SI1005 appear tobe more potent inhibitors of MMP enzymes and this may be an advantage inthe management of cardiovascular disease and cancer. SI1004 appears tobe more MMP-9 specific and does not reduce MMP-3 in in vitroinflammatory cell models. Both nitrocyclines SI1004 and SI1005 are moreeffective immunomodulatory compounds. They do not appear to activateMMP-2, unlike high concentration (100 μM) doxycycline. Finally, they aremore effective in reducing tumour cell invasiveness in an in vitro modelwith human colon cancer cells.

In Vivo, Non-Human Primate Study. Methods

Purpose bred, naïve, non-human primates (cynomolgus monkeys, 2.9-4 kg)were sourced and randomly allocated in a parallel group design (n=6 pergroup) to receive SI1004, SI1005 and equimolar doses of doxycyclinedaily (1.6 mg/kg doxycycline hyclate equivalents, on day 1 and 4.8 mg/kgdoxycycline equivalents on days 2 and 3) by oral gavage in aqueousvehicle over a 3 day period. Studies were carried out consecutively intwo contract research organization sites (Charles River, Sparks, Nev.,US and Charles River, Shanghai, China). The study protocol was approvedby PCS-SHG Institutional Animal Care and Use Committee before conduct.During the study, care and use of animals was conducted in accordancewith the guidelines of the USA National Research Council and theCanadian Council on Animal Care. The cynomolgus monkey was chosen forthis study in order to maximize the likelihood of identifying responsesthat are similar to those that may be expected in humans. Each animalwas identified by a cage label and body tattoo and was acclimated toorogastric dosing on at least two occasions prior to the initiation ofdosing. The vehicle (1% (w/v) tween 80 and 0.5% (w/v)carboxymethylcellulose in deionized water) or 1.6 mg/kg doxycyclinehyclate (0 hours) or 4.8 mg/kg doxycycline hyclate (24, 48 hours) or themolar equivalent(s) of SI1004 or SI1005 were administered using anorogastric tube inserted through the mouth and advanced into thestomach. The animals were temporarily restrained (i.e. manually) fordose administration, and were not sedated. Disposable sterile syringesand orogastric tubes were used for each animal/dose. Each dose wasfollowed by a tap water flush of approximately 5 mL. Blood samples andblood pressure measurements were taken at the following timepoints:pre-dose (0 hours) and at 2, 4, 6, 12, 24, 26, 30, 36, 48, 50, 54, 60and 72 hours after first administration of test article. Blood (300 μL)for serum preparation was collected intoBD Vacutainer®+Serum SST™ tubesto accelerate clotting 20 minutes prior to centrifugation to allowcomplete clotting to occur and centrifuged at 1500-2200 rpm at 2-8° C.for 10-15 minutes. Under these conditions blood cells containing MMP,principally neutrophils and platelets, undergo full degranulation. Sinceartifactual elevation of MMP-9 was an unavoidable feature of repeatedvenipuncture in our model, it was logical to stimulate full MMP-9release during sample collection. This provided greater inter-animalreproducibility and a more dynamic analytical range for assessing therelative effects of the test articles. Subsequent MMP-9 values providean index of total MMP-9 including circulating enzyme, amplified byrepeated venipuncture, along with the cellular load released fromstorage granules during clotting. The latter is influenced by earlierinflammatory signaling, transcription and storage. The serum wastransferred to a cryovial and immediately stored at −70° C. untilanalyzed for MMP-2 and MMP-9 via a Luminex ELISA (total MMP-2 and MMP-9)within 48 hours of collection. The analysis of each time point wasrepeated within 5 days. Values that differed by more than 15% wererepeated. The primary study endpoint was the change in plasma MMP-2 andMMP-9 levels at 72 hours. Secondary endpoints were area under the curve(AUC) values of MMP-2 and MMP-9 over the following periods: 0-24, 0-48and 0-72 hours.

Data on Admixtures (FIGS. 13-17)

Admixtures of tetracyclines and nitric oxide donors have benefits ininflammatory and cardiovascular diseases. In FIG. 13, Doxy and nitrate Aadmixture (Diethanolamine dinitrate, the alkyl nitrate component ofSI1004) are significantly better than Doxy at inhibiting cardiacfibroblast proliferation (p=0.011) at 150 micromolar. However, Doxy andnitrate B admixture (Nitroxymethyl piperidine) are not (p=NS) at sameconcentration. The novel nitrocycline, SI1004, is significantly moreeffective at inhibiting cardiac fibroblast proliferation thandoxycycline, Doxy and nitrate A admixture, Doxy and nitrate B admixtureat 150 micromolar (all p<0.01). In some cases, inflammatory cytokinesare similarly reduced by Doxy and admixtures with NO donors. In FIG. 14,Doxy and nitrate A admixtures are shown to significantly reduce IL-8levels in TNFalpha stimulated PBMCs at 150 micromolar (p<0.01). Doxyalone and Doxy and nitrate B admixtures also reduce IL-8 levels comparedto controls (p<0.05). However, in some instances, the effects Doxy andnitrate A admixture is more effective than Doxy. In FIG. 15, Doxy andnitrate A (Diethanolamine dinitrate) admixture can significantly reduceIL-1 beta levels in TNFalpha stimulated PBMCs (p<0.05). Doxy and nitrateB (Nitroxymethyl piperidine) admixture reduce IL-1 beta levels, but notsignificantly (p=NS). Furthermore, in some instances, the choice of NOdonor dramatically alters the anti-inflammatory effects. In FIG. 16 itis shown that IL-4 is reduced significantly more (p<0.01) by Doxy andnitrate A (Diethanolamine dinitrate) admixture than either Doxy or Doxyand nitrate B (Nitroxymethyl piperidine) admixture. IL-4 is implicatedin inflammatory bowel disease. IL-8 is implicated in invasive bladdercancer, chronic prostatitis, acute pyelpnephritis, non-Hodgkinslymphoma, pulmonary infections and osteomyelitis. IL-1β is implicated infever, anemia, cryopyrinopathies (hereditary periodic fever syndromes),gout and pseudogout, Septic shock.

The invention claimed is:
 1. A compound selected from:amido-N-[3-methylnitratepiperidinomethy]-α-6-deoxy-5-oxytetracyclineamido-N-[N,N-diethylnitrate-aminomethyl]-α-6-deoxy-5-oxytetracycline(amido-N-[bis-(β-nitrooxyethyl)aminomethyl]-α-6-deoxy-5-oxytetracycline)amido-N-[(β-nitrooxyethyl)aminomethyl]-α-6-deoxy-5-oxytetracyclineamido-N-[3-(nitrooxymethyl)piperidinomethyl]-α-6-deoxy-5-oxytetracyclineamido-N-[3-(nitrooxymethyl)piperidinomethyl]-α-6-deoxy-5-oxytetracyclineamido-N-[4-(nitrooxymethyl)piperidinomethyl]-α-6-deoxy-5-oxytetracyclineamido-N-[4-nitrooxypiperidinomethyl]-α-6-deoxy-5-oxytetracyclineamido-N-[4-nitrooxypiperidinomethyl]-tetracyclineamido-N-[bis-(β-nitrooxyethyl)methylaminomethyl]-β-6-deoxy-5-oxytetracyclinamido-N-[bis-(β-nitrooxyethyl)methylaminomethyl]-α-6-deoxy-5-oxytetracyclinamido-N-[bis-(β-nitrooxyethyl)ethylaminomethyl]-tetracyclineamido-N-[(β-nitrooxyethyl)aminomethyl]-tetracyclineamido-N-[4-(nitrooxymethyl)piperidinomethyl]-tetracycline oramido-N-[3-(nitrooxymethyl)piperidinomethyl]-tetracycline.
 2. Thecompound according to claim 1 selected from the group consisting of:


3. A compound selected from the group consisting of:


4. A pharmaceutical composition comprising a compound according to anyone of claims 1, 2, and 3 and a carrier.